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BACTERIOLOGICAL  METHODS 


SCHNEIDER 


BY  THE  SAME  AUTHOR 

PHARMACEUTICAL  BACTERIOLOGY 

80   Illustrations 

Octavo  246  Pages 

Cloth,  $2.00  Post  Paid 


"The  discussion  of  disinfectants  and  of  the  principles 
of  disinfection  and  sterilization  and  of  the  practical 
application  of  these  principles  in  the  pharmacy  would 
alone  make  the  book  well  worth  while  to  every  phar- 
macist."— Jnl.  Amer.  Pharmaceutical  Ass'n. 


Bacteriological  Methods 

IN 

Food  and  Drugs  Laboratories 

WITH  AN 

Introduction  to  Micro-analytical  Methods 


BY 

ALBERT  SCHNEIDER,  M.  D.,  Ph.  D, 

^'(Columbia  University), 

PROFESSOR  OF  PHARMACOGNOSY  AND  BACTERIOLOGY   IN  THE 

COLLEGE   OF    PHARMACY   OF   THE    UNIVERSITY   OF 

CALIFORNIA,   SAN  FRANCISCO 


87  ILLUSTRATIONS 
AND  6  FULL  PAGE  PLATES 


PHILADELPHIA 

P.   BLAKISTON'S   SON   &   CO. 

1012  WALNUT   STREET  • 


TK 


r  - 
3Sb5 


Copyright,  1915,  by  P.  Blakiston's  Son  &  Co. 


'THK     MAPLE     PRKSS     YORK     PA 


PREFACE 


The  administration  of  the  Federal  Pure  Food  and  Drugs  Act 
and  of  the  several  State  Pure  Food  and  Drugs  Laws  has  made  the 
introduction  of  bacteriological  methods  into  food  and  drugs 
laboratories  a  necessity.  Because  of  the  close  relationship  be- 
tween the  work  of  the  bacteriologist  and  that  of  the  micro-analyst, 
it  is  advised  that,  wherever  possible,  these  two  laboratory  branches 
be  combined  in  the  most  efifectual  cooperative  manner.  With 
such  cooperation  in  mind,  a  brief  introduction  to  micro-analytical 
methods  is  added.  Fuller  details  on  micro-technique  will  be 
found  in  special  works  on  the  microscopy  of  fibers,  foods,  spices, 
drugs,  of  water  supplies,  of  sewage,  etc. 

As  is  more  fully  set  forth  in  the  text,  the  bacteriological  as 
well  as  micro-analytical  methods  in  our  food  and  drugs  labora- 
tories are  not  yet  fully  worked  out,  and  the  present  volume  is 
submitted  hoping  that  it  will  be  instrumental  in  bringing  about 
a  unification  of  methods  and  that  it  will  perhaps  also  serve  as  a 
guide  to  the  working  out  of  newer  and  inadequately  tested  older 
methods. 

The  volume  is  primarily  intended  as  a  guide  to  students  who 
are  interested  in  the  bacteriological  examination  of  foods  and 
drugs.  Its  use  as  a  laboratory  guide  presupposes  a  thorough 
knowledge  of  general  bacteriology. 

Acknowledgments  are  made  to  the  following  authors  for  the 
use  of  illustrations:  E.  R.  Stitt  (Bacteriology,  Blood  Work  and 
Animal  Parasitology),  R.  L.  Pitfield  (Compend  on  Bacteriology), 
W.  J.  MacNeal  (Pathogenic  Micro-organisms),  C.  E.  Marshall 
(Microbiology),  John  F.  Anderson  and  Thomas  B.  McClintic 
(Method  of  Standardizing  Disinfectants),  and  G.  W.  Hunter  (Es- 
sentials of  Bioloery).  * 

333721 


VI  PREFACE 

Grateful  acknowledgments  are  also  made  to  A.  E.  Graham, 
Inspector  in  Charge,  San  Francisco  Laboratory,  Bureau  of  Animal 
Industry,  for  valuable  suggestions  regarding  the  examination  of 
meats  and  meat  products  with  special  reference  to  the  isolation 
and  examination  of  animal  fat  crystals  and  the  examination  of 
sausage  meats  for  'starch  fillers;  to  Professor  Karl  Frederick 
Meyer,  of  the  Department  of  Bacteriology  and  Protozoology  of  the 
University  of  California,  for  the  article  on  "The  Precipitin  Test 
for  Meats,"  and  to  Merck's  Report  for  permission  to  use  those 
parts  of  the  text  which  had  been  published  in  that  journal.  It  is 
also  desired  to  acknowledge  the  loan  of  several  cuts  by  the  Bausch 
and  Lomb  Optical  Company.  Additional  acknowledgments  are 
made  throughout  the  text. 
San  Francisco,  California. 


CONTENTS 


I.  Outline  of  Micro- analytical  Methods  in  Food  and  Drugs  Laboratories 

Page 

1.  Introduction i 

2.  Grouping  of  Substances  to  be  Examined  in  Food  and  Drugs  Laboratories .    .  i 

3.  The  Work  of  the  Micro-analyst  in  Relationship  to  that  of  the  Chemist  and 

Bacteriologist 3 

4.  Equipment  for  Micro-analytical  Work 4 

5.  Organoleptic  Testing 8 

6.  Methods  Useful  in  the  Examination  of  Vegetable  Drugs,  Spices,  Etc  ...  9 

7.  Methods  Useful  in  the  Examination  of  Vegetable  Food  Products    ....  n 

8.  Micro-chemical  Color  Reaction  Tests 15 

9.  Making  Analytical  Reports  .    .    .    '. 17 

10.  The  More  Important  Histological  Elements  of  Plants 18 


11.  Bacteriological  Methods  in  Food  and  Drugs  Laboratories 

1.  Introduction 23 

2.  Direct  Bacteriological  Examinations 51 

3.  Numerical  Limits  of  Micro-organisms  in  Foods  and  Drugs 53 

4.  Quantitative  Estimations  by  the  Cultural  Methods 68 

5.  Preparation  of  Standard  Culture  Media.     General  Suggestions    .....  71 

6.  Preparation  of  Required  Standard  Culture  Media 75 

7.  Technique  for  Making  Quantitative  and  Qualitative  Estimations  by  the 

Plating  Methods 83 

8.  Practical  Application   of  the   Quantitative  Estimations  by  the  Plating 

Methods 89 

9.  Qualitative  Determinations 90 

10.  Evidence  of  Sewage  Contamination 97 

11.  Possible  Contamination  of  Foods  with  the  Typhoid  Bacillus 102 

12.  Possible  Contamination  of  Food  Substances  with  the  Cholera  Bacillus.    .    .112 

13.  Biological  Water  Analysis 114 

14.  Bacteriological  Examination  of  Mineral  Waters 118 

15.  The  Microscopical  and  Bacteriological  Examination  of  Milk 120 

16.  The  Bacteriological  Examination  of  Shellfish 144 

17.  The  Bacteriological  and  Toxicological  Examination  of  Meats  and  Meat 

Products 152 

vii 


vm  CONTENTS 

Page 

1 8.  The  Bacteriological  Examination  of  Eggs  and  Egg  Products 187 

19.  The  Bacteriological  Examination  of  Pharmaceutical  Preparations    ....   197 

20.  The  Microscopical  and  Bacteriological  Examination  of  Syrups 202 

21.  The  Microscopical  and  Bacteriological  Examination  of  Fermented  Foods 

and  Beverages 210 

22.  Standardization  of  Disinfectants .....    230 

23.  Determining  the  Purity  and  Quality  of  Sera,  Bacterins  and  Related  Products  265 

24.  Special  Biological  and  Toxicological  Tests 268 


OUTLINE  OF  MICRO-ANALYTICAL  METHODS  IN 
FOOD  AND  DRUGS  LABORATORIES 


I.  Introduction 


The  value  of  the  compound  microscope  in  the  examination  of 
foods  and  drugs  is  as  yet  not  generally  recognized.  Efficiency  in 
this  line  of  work  depends  very  largely  upon  a  long  and  wide  range 
of  experience,  in  this  regard  differing  very  markedly  from  effi- 
ciency in  the  field  of  chemical  analyses.  All  that  is  required  of 
the  chemist,  as  far  as  routine  analytical  work  is  concerned,  is  a 
very  close  adherence  to  the  methods  laid  down  for  him.  He  is 
pronounced  skilled  in  direct  proportion  to  his  adherence  to 
methods  and  skill  shown  in  the  manipulation  of  apparatus  and 
reagents.  The  micro-analyst  in  order  to  be  efficient  must  be  very 
familiar  with  the  appearance  of  the  multitudinous  forms  of  cells, 
tissues,  cell-contents  and  with  the  behavior  of  certain  micro- 
chemical  reagents  and  this  familiarity  can  be  acquired  only  through 
long  and  careful  observation. 

2.  Grouping  of  Substances  to  be  Examined  in  Food  and  Drugs 

Laboratories 

The  analytical  methods,  as  they  apply  to  the  critical  examina- 
tion of  foods  and  drugs,  are  chemical,  microscopical  and  bacteriolog- 
ical.    The  substances  to  be  analyzed  may  be  grouped  as  follows: 


2  MICRO-ANALYTICAL  METHODS 

1.  Vegetable  drugs,  crude  and  powdered,  pharmacopceial  and  other  simple  and 
compound  medicinal  powders. 

2.  Spices  and  condiments,  whole,  ground  and  powdered.  Prepared  spices  and 
condiments. 

3.  Coffee,  tea,  cocoa,  chocolate,  confections,  candies. 

4.  Tobacco  and  preparations  made  from  tobacco,  as  snuff,  smoking  tobacco, 
cigars,  etc. 

5.  Chemicals,  minerals,  solutions  of  chemicals,  etc. 

6.  Tablets,  pills,  powders. 

7.  Meats  of  all  kinds,  raw,  cooked,  canned,  sausage  meats,  etc. 

8.  Dairying  products,  as  milk,  cream,  cheese,  butter,  ice-cream,  ice  cream  fillers, 
etc. 

9.  Insect  powders,  dusting  powders,  cosmetics. 
ID.  Cattle  and  poultry  powders. 

11.  Unknown  powders,  wholly  or  partly  of  vegetable  origin. 

12.  Starches,  dextrins,  sausage  meat  binders  (starches). 

13.  Vegetable  foods,  as  jams  and  jellies;  fresh,  pickled,  cooked,  canned  and 
preserved. 

14.  Flours  and  meals. 

15.  Breakfast  foods,  infant  and  invalid  foods. 

16.  Breads    and    similar  materials;  biscuits,  doughnuts,  cakes,  pies,  pastries, 
etc. 

17.  Macaroni,  spaghetti  and  similar  preparations,  noodles,  etc. 

18.  Nuts  and  nut-like  fruits  and  seeds,  etc. 

19.  Beverages  of  all  kinds,  liquids  generally. 

20.  Pharmaceuticals  of  all  kinds. 

21.  Patent  and  proprietary  medicines. 

22.  Unknown  foods  and  medicines. 

In  the  examination  of  some  of  these  substances  the  chemical 
method  is  all  important,  as  in  chemicals  generally;  in  the  examina- 
tion of  others  the  microscopical  method  is  all-important,  as  in 
meals,  flours,  spices;  and  again  the  bacteriological  testing  is  all- 
important,  as  in  sewage,  contaminated  water,  contaminated  milk, 
infected  foods  and  drinks  generally,  etc.  A  properly  equipped 
analytical  laboratory,  whether  federal,  state  or  private,  should 
be  prepared  to  apply  all  three  methods.  The  bacteriological  in- 
vestigations should  be  made  by  the  micro-analyst  rather  than  by 
the  chemist,  because  of  the  closer  relationship  between  bacteriology 
and  microscopy.. 


INTRODUCTION  3 

3.  The  Work  of  the  Micro -analyst  in  Relationship  to  that  of  the 
Chemist  and  Bacteriologist 

Just  what  work  should  or  should  not  be  done  by  the  micro- 
analyst  is  as  yet  not  definitely  determined;  at  least,  there  is  no 
uniformity  as  to  scope  of  action  in  the  different  analytical  labora- 
tories. It  is  suggested  that  the  following  work  be  assigned  to  the 
micro-analyst: 

1.  Gross  and  net  weight  determination  of  all  such  samples  as  require  it. 

2.  Moisture  determination  of  substances  which  require  it. 

3.  Ash  and  acid  insoluble  determinations  of  substances  which  are  primarily 
subject  to  microscopical  analysis,  as  vegetable  drugs,  pills,  powders,  vegetable 
compound  powders,  etc. 

4.  Use  of  certain  special  tests,  as  sublimation  tests  for  benzoic  acid,  salicylic  acid 
and  boric  acid;  Grahe's  cinchona  test,  wheat  gluten  test,  color  reactions  for  boric 
acid,  capsicum,  guaiac,  salicylic  acid,  morphine,  etc.,  tests  for  cholesterol  and  phy- 
tosterol  crystals,  and  others  which  may  prove  useful. 

5.  Bacteriological  testing  of  foods  and  drugs  generally,  of  sera,  vaccines,  galen- 
icals, syrups,  milk,  water,  jams,  jellies,  catsups,  etc.,  as  may  be  required,  following 
the  method  of  the  Society  of  the  American  Bacteriologists,  and  limiting  the  testing 
to  determining  the  presence  or  absence  of  the  colon  bacillus  and  other  sewage  organ- 
isms, and  the  usual  quantitative  bacterial  determinations  for  milk,  water  and  other 
substances,  of  which  the  quality  is  usually  based  upon  the  quantitative  bacterial 
content. 

Substances  subject  to  analysis  in  the  laboratories  mentioned 
should  be  grouped  or  classified  according  to  the  special  or  pre- 
ferred methods  of  examination  to  be  applied.  It  is,  of  course, 
evident  that  in  the  majority  of  cases  chemical  as  well  as  micro- 
scopical methods  should  be  used.  In  some  cases  even  all  three 
must  be  used  in  order  that  conclusive  results  may  be  obtained. 
The  following  grouping  is  suggested: 

1.  Substances  in  which  the  chemical  analysis  is  of  first  importance.  Chemicals 
generally,  and  chemicals  in  solution,  alcohol,  alcoholic  drinks,  flavoring  extracts, 
syrups,  oils,  fats,  etc. 

2.  Substances  in  which  the  microscopical  analysis  is  of  first  importance — 
vegetable  substances  and  preparations  which  are  essentially  of  vegetable  origin. 
Meats  of  all  kinds,  variously  prepared,  cooked,  spiced,  etc. 

3.  Substances  in  which  the  chemical  and  microscopical  examinations  are  of  equal 


4   .  MICRO-ANALYTICAL   METHODS 

importance — assayable  vegetable  drugs,  all  prepared  food  substances  with  chemicals 
in  solution,  compound  powders,  pills,  tablets. 

4.  Substances  to  which  the  microscopical  examination  is  not  generally  applied 
■ — chemicals,  liquids  in  which  the  insoluble  particles  are  slight  in  amount,  as  wines, 
brandies,  comparatively  pure  solutions,  etc.  Here  the  centrifuge  plays  an  im- 
portant part. 

5.  Substances  in  which  the  bacterial  testing  is  of  prime  importance — milk, 
sewage  or  otherwise  organically  contaminated  water  supplies,  and  other  liquids, 
beers,  etc.,  contaminated  foods  generally.  In  this  class  of  substances  the  micro- 
scopical and  chemical  examinations  become  necessary  in  addition  to  the  bacterio- 
logical; in  fact,  a  bacteriological  test  is  incomplete  without  the  use  of  a  good  com- 
pound microscope. 

The  work  of  the  micro-analyst  is,  so  to  speak,  on  trial.  The 
doubt  in  the  minds  of  the  critics  is  due,  very  largely,  to  the  un- 
satisfactory results  traceable  to  the  efforts  of  those  who  are  not 
sufficiently  qualified.  Even  the  most  skillful  analysts  admit 
numerous  defects  in  methods  and  shortcomings  in  results.  For 
example,  the  quantitative  estimates  based  upon  optical  judg- 
ment are  approximate  only,  and  with  most  workers  there  is  a 
very  marked  tendency  to  make  these  estimates  volumetric  rather 
than  gravimetric.  This  can  in  a  measure  be  corrected  by  bring- 
ing into  play  the  judgment  of  the  relative  weights  of  the  several 
substances  under  comparison.  For  example,  the  amount  of 
sand  present  in  powdered  belladonna  root  may  be  volumetrically 
estim.ated  at  20  per  cent.  In  this  case  the  acid  insoluble  ash 
residue  may  show  35  to  40  per  cent,  of  silica.  An  example  like 
this  also  indicates  why  the  micro-analyst  should  make  the  sand 
and  ash  determinations.  The  percentage  estimates  based  upon 
microscopical  examination  may  vary  within  25  to  50  per  cent, 
when  the  amounts  of  the  admixtures  are  small  or  slight.  For 
example,  the  actual  amount  of  arrow-root  starch  in  the  so-called 
arrowroot  biscuit  is  2.5  per  cent.  The  micro-analyst's  estimates 
may  range  from  a  trace  or  small  amount  to  5  per  cent.  When 
the  quantities  of  admixtures  are  large,  from  30  to  90  per  cent., 
the  estimations  may  approximate  within  10  or  15  per  cent,  of 
the  actual  amount  present.     These  estimates  can  no  doubt  be 


INTRODUCTION  5 

made  much  more  accurate  by  uniform  methods  of  technique, 
aided  by  certain  mechanical  devices.  For  example,  in  the  ex- 
amination of  vegetable  powders,  spices,  meals,  flours  and  similar 
substances,  the  samples  should  be  thoroughly  mixed,  and  slide 
mounts  should  be  of  standard  and  uniform  thickness  and  the 
relative  amounts  of  the  ingredients  should  be  estimated  by  means 
of  microscope  slides  having  uniform  ruled  squares  of  definite 
measuring  value  in  microns.  These  and  other  details  in  the 
methods  should  be  more  fully  worked  out. 

Several  micro-analysts  have  declared  themselves  as  opposed 
to  giving  percentage  estimates  of  the  several  ingredients  of  a 
compound.  However,  not  to  give  the  approximate  percentages 
will  cause  great  confusion  and  very  materially  lessen  the  value  of 
the  work  done.  For  example,  to  report  a  pancake  flour  as  com- 
posed of  ''buckwheat  and  wheat  flour,  the  former  predominating," 
instead  of  "buckwheat  approximately  75  per  cent,  and  wheat 
approximately  25  per  cent.,"  would  certainly  be  unsatisfactory. 

The  following  examples  will  serve  to  explain  the  relative  value 
of  the  chemical  and  microscopical  analyses.  Suppose  the  sub- 
stance to  be  examined  is  a  baby  food.  The  microscope  may  re- 
veal approximate  percentages  of  oil  globules,  steam  dextrinized 
wheat  starch,  unchanged  wheat  and  arrowroot  starch,  wheat 
tissue  and  milk  sugar.  The  chemical  analysis  will  show  a  definite 
percentage  of  sugar,  soluble  starch,  insoluble  starch,  fat,  vege- 
table fiber  and  ash.  This  is  a  good  example  of  a  case  where  the 
two  methods  of  analysis  are  of  equal  importance;  one  without 
the  other  would  be  unsatisfactory,  incomplete  and  inconclusive. 
Again,  the  chemical  assay  may  show  that  a  sample  of  powdered 
belladonna  leaf  contains  0.35  per  cent,  of  mydriatic  alkaloids,  and 
yet  the  microscopical  examinations  may  prove  the  presence  of 
20  per  cent,  or  more  of  some  foreign  leaf. 

An  adjunct  in  analytical  work,  much  neglected  by  the  chemist, 
is  the  organoleptic  testing.  This  is  especially  important  in 
the  examination  of  unknown  substances,  fruit  products,  spices, 


6  MICRO-ANALYTICAL  METHODS 

meats,  etc.,  as  it  often  gives  a  clue  to  the  quality  of  the  sub- 
stances and  to  the  means  of  getting  quick  results. 

4.  Equipment  for  Micro -analytical  Work 

The  equipment  and  apparatus  required  by  the  micro-analyst 
is  comparatively  inexpensive,  and  it  is  very  earnestly  advised  to 
secure  only  those  appliances  which  are  useful  or  essential  for  the 
work  in  hand.  The  following  list  is  submitted  without  entering 
into  detail,  as  it  may  be  assumed  that  the  microscopist  does  not 
require  explanations : 

1.  Simple  lens. 

2.  Compound  microscope. 

a.  Ocular  with  micrometer  scale. 
h.  Oculars,  Nos.  2  and  4. 

c.  Objectives,  Nos.  3,  5  and  7. 

d.  1/12  in.  oil-immersion  objective  for  bacteriological  work. 

3.  Slides  and  covers. 

4.  Section  knife  or  razor,  and  strop. 

5.  Polarizer,  for  the  study  of  starches,  crystals  and  other  substances.  Should 
be  convenient  to  use.     The  selenite  plates  are  useful. 

6.  Thoma-Zeiss  hemacytometer;  for  counting  bacteria  and  yeast  cells. 

7.  Stage  mold  and  spore  counter,  as  described  in  Part.  II  (Fig.  5). 

8.  Accurate  metal  or  hard  rubber  millimeter  ruler  for  measuring  seeds  (in  fruit 
products),  etc. 

9.  The  required  glassware  and  adjunct  apparatus. 

10.  The  required  reagents. 

11.  Equipment  for  making  moisture  determinations. 

12.  Equipment  for  making  ash  determinations. 

13.  Equipment  for  the  required  bacteriological  tests  and  determinations. 

The  laboratory  in  which  the  work  is  done  should  be  roomy,  well- 
lighted,  provided  with  the  necessary  shelves,  apparatus  and  supply 
cases,  reference  books,  etc.  The  details  need  not  be  given  here. 
The  analyst  must  see  to  it  that  the  necessary  things  are  provided. 
A  skillful  and  experienced  worker  should  have  the  tools  of  his 
choice,  not  those  selected  for  him  by  some  one  not  qualified  to 
judge. 

The  skilled  micro-analyst  has  little  difficulty  in  determining 


LABORATORY  EQUIPMENT  7 

the  purity  and  comparative  quality  of  the  simple  spices,  as  pepper, 
allspice,    cloves,    cinnamon   and   ginger.     However  matters   are 


Fig.  I. — Form  of  compound  microscope  suitable  for  bacteriological  and  general 
microscopical  work  in  food  and  drugs  laboratories.  Note  the  desirable  and  necessary 
accessories  as  given  in  the  text.  The  form  of  polarizing  apparatus  convenient  to  be 
used  with  the  compound  microscope,  sets  into  the  substage  diaphragm  ring  with  the 
iris  diaphragm  opened  to  the  maximum.  The  analyzer  takes  the  place  of  the  ocular. 
— (Bausch  &•  Lomb  Co.) 


quite  different  when  it  comes  to  the  examination  of  powdered 
vegetable  drugs,  compound  vegetable  powders  and  vegetable 
products  of  unknown  composition.     A  thorough  knowledge  of, 


8  MICRO-ANALYTICAL   METHODS 

and  a  wide  familiarity  with,  cell-forms,  tissue  elements  and  formed 
cell  contents  is  an  absolute  essential  in  order  that  accurately  re- 
liable and  conclusive  results  may  be  obtained  and  serious  con- 
fusion may  be  avoided.  Differences  in  the  reports  of  findings  by 
micro-analysts  are  in  part  due  to  the  personal  equation,  in  part 
due  to  variable  methods  and  differences  of  judgment  in  estimat- 
ing the  quantity  of  tissue  elements  present  and  also  in  part  due 
to  a  lack  of  extensive  and  intensive  experience. 

5.  Organoleptic  Testing 

The  organoleptic  tests  are  indeed  valuable  adjuncts  to  the  micro- 
scopical work.  There  are,  however,  some  differences  of  opinion 
regarding  the  interpretation  and  valuation  which  ai  e  to  be  placed 
on  comparisons  of  color,  odor  and  taste,  even  among  those  having 
had  considerable  experience  and  endowed  with  a  fairly  normal 
special  sense  development.  Our  color  terminology  is  in  great 
confusion,  and  so  far  as  the  olfactory  sense  is  concerned,  there 
are  only  comparatively  few  odors  or  flavors  which  admit  of  ready 
comparison  such  as  tea  flavor,  coffee  odor,  vanilla  odor,  raspberry 
flavor,  loganberry  flavor,  and  the  odor  of  such  drugs  as  valerian, 
cubeb,  fenugreek,  asafetida,  aloes,  turpentine,  camphor,  the  essen- 
tial oils,  calamus,  etc.,  and  the  odor  of  the  spices.  Our  compara- 
tive judgment  of  tastes  is  more  reliable.  Much  experience  is 
necessary  to  form  fairly  reliable  estimates  of  flavors  (associations  of 
tastes  and  odors),  though  pure  fruit  flavors  are,  as  a  rule,  readily 
distinguishable,  as  that  of  apples,  dried  apples,  peach,  dried  peach, 
quince  and  strawberry.  Manufactured  fruit  preparations  gener- 
ally lose  much  of  their  flavor  due  to  many  causes,  as  cooking, 
steaming,  fermentative  changes,  presence  of  decayed  (moldy) 
fruits,  mixing  of  several  kinds  of  fruits  or  fruit  juices,  etc.,  to  say 
nothing  of  the  wholly  artificial  or  imitation  fruit  flavors  and  the 
flavors  of  the  imitation  fruit  products  which  have  little  or  no  fruit 
in  their  composition. 


SPECIAL    TESTS  9 

6.  Methods  Useful  in  the  Examination  of  Vegetable  Drugs, 
Spices,  Etc. 

We  shall  give  a  few  tests  which  have  proven  useful  in  the  ex- 
amination of  drugs  and  food  products.  It  will  be  found  that 
many  of  the  test  results  are  largely  approximate,  and  some  of 
them  are  primarily  intended  to  serve  as  aids  or  checks  to  the 
chemical  examination. 

1.  Mace  Test. — To  a  pinch  of  the  powdered  mace  add  a  few 
drops  of  10  per  cent,  sodium  hydroxide  solution.  Banda  or  true 
mace  changes  color  only  slightly,  whereas  wild  or  Bombay  mace 
turns  a  deep  orange  color. 

2.  Conium  Test. — To  the  substance  to  be  tested  for  the  presence 
of  conium  fruits  (as  anise,  caraway  or  other  umbelliferous  fruits), 
add  25  per  cent,  sodium  or  potassium  hydroxide  solution.  In 
the  presence  of  i  per  cent,  or  more  of  conium  fruits  a  distinct 
mouse  odor  is  developed  in  time  (lo  min.  to  Yi  hr.).  This  test 
is  not  reliable  with  old  umbelliferous  fruits,  as  many  of  them  de- 
velop a  more  or  less  marked  mouse  odor  with  alkalies. 

3.  Lignin  Test. — The  classic  phloroglucin-hydrochloric  acid  test 
is  usefyl  in  making  estimates  of  the  amount  of  lignified  tissue 
present,  as  in  old  belladonna  root,  aconite  roots  and  stems, 
lobelia  herb,  fruit  products,  spices,  etc. 

4.  Grahe's  Cinchona  Test. — Drive  the  moisture  from  the  inner 
surface  of  a  small  test-tube  by  holding  it  over  a  Bunsen  burner. 
Into  this  dried  test-tube  place  a  pinch  of  finely  powdered  cinchona 
bark  (No.  80)  and  heat  rather  carefully  over  an  alcohol  lamp  or 
Bunsen  burner.  When  the  bark  begins  J:o  char,  red  fumes  begin 
to  fill  the  tube  and  condense  on  the  side  of  the  tube  as  a  reddish 
purplish  liquid.  The  intensity  of  the  reaction  is  approximately 
proportional  (direct  proportion)  to  the  percentage  of  alkaloids 
present.  Some  skill  and  experience  is  necessary  to  perform  this 
test  well.  The  tube  must  not  be  heated  too  quickly  or  too  much, 
and  the  powder  should  be  uniformly  fine. 


lO  MICRO-ANALYTICAL  METHODS 

5.  Beaker  Sand  Test. — Pour  a  definite  amount  of  the  powdered 
spice  or  vegetable  drug  into  a  beaker,  add  water,  stir  until  the 
sand  is  washed  away  from  the  vegetable  particles  and  settles  to 
the  bottom  of  the  beaker.  Let  a  stream  of  water  run  into  beaker 
so  as  to  wash  out  the  vegetable  matter.  The  final  washing  and 
decanting  must  be  done  carefully  so  as  not  to  lose  the  sand.  Salt 
brine  may  be  used  instead  of  water,  should  the  vegetable  matter 
have  a  comparatively  high  specific  gravity.  Dry  the  sand  and 
weigh  to  obtain  the  percentage  of  sand  present. 

6.  Ash  Determination. — According  to  the  regulation  method. 
The  percentage  of  the  acid-insoluble  residue  should  also  be  de- 
termined. It  should  be  borne  in  mind  that  the  ash  determination 
gives  only  approximate  results  as  far  as  the  presence  of  clay  and 
dirt  is  concerned,  since  the  organic  matter  of  dirt  is  combustible. 
The  ash  percentage  varies  greatly  in  vegetable  drugs,  especially 
in  herbs  and  leaves.  The  sand  percentage  is  comparatively  high 
in  those  herbs  and  leaves  having  abundant  trichomes,  especially 
if  the  drug  plants  (or  herbaceous  spices)  bearing  such  trichomes  are 
grown  in  dry  sandy  soil.  Dirt  (and  sand)  percentage  is  apt  to 
be  high  in  roots  and  rhizomes,  particularly  when  rootlets  are 
abundant  and  when  the  gathering,  garbling  and  cleaning  is 
carelessly  done. 

There  are  a  number  of  chemical  tests  giving  color  reactions 
which  can  be  done  conveniently  by  the  micro-analyst,  as  the  boric 
acid  reaction  with  curcuma,  the  H2SO4  color  reaction  with  some 
barks,  capsicum,  guaiac,  resin,  cubeb,  etc.;  the  H2SO4  plus  for- 
maldehyde color  reaction  with  morphine;  the  ferric  chloride 
color  reaction  with  salicylic  acid,  etc.  These  tests  should  be 
used  when,  in  the  judgment  of  the  analyst,  they  may  serve  to 
give  better  information  regarding  the  identity,  purity  and  quality 
of  the  drug. 


SPECIAL  TESTS  II 

7.  Methods  Useful  in  the  Examination  of  Vegetable  Food 

Products 

1.  Sublimation  Test  for  Benzoic  Acid. — Place  a  drop  or  two 
of  the  suspected  liquid  or  semi-liquid  food  substance  into  a 
deep  watch  crystal  of  i  in.  diameter.  Place  over  it  a  clean  dry 
slide.  Now  hold  the  watch  crystal  over  a  flame  (alcohol  lamp^) 
until  the  substance  (as  wine,  vinegar,  catsup,  jam,  jelly,  etc.), 
comes  to  an  active  boil.  The  steam  vapor,  carrying  with  it 
the  benzoic  acid,  is  condensed  on  the  slide.  Remove  the  slide 
and  set  it  aside  until  the  condensed  moisture  has  evaporated;  very 
moderate  heat  may  be  used  to  hasten  evaporation.  Examine 
under  the  microscope,  whereupon  the  benzoic  acid  crystals  may 
be  seen,  provided  any  were  present.  The  test  is  delicate,  very 
reliable  and  very  few  substances  interfere  with  it.  It  is  very 
pronounced  in  the  presence  ofo.oi  per  cent,  of  benzoic  acid. 

2.  Sublimation  Test  for  Salicylic  Acid.^ — Made  like  the  benzoic 
acid  test.  The  crystal  formation  (plates)  is  very  pronounced  in 
dilutions  of  i  :  looo.  After  having  examined  the  crystals  under 
the  microscope,  add  a  drop  of  weak  solution  of  ferric  chloride  to 
the  crystals  upon  the  slide,  whereupon  a  blue  coloration  develops. 
Boric  acid  is  likewise  deposited  by  sublimation,  but  the  test  is 
not  as  satisfactory  as  those  for  benzoic  acid  and  for  salicylic  acid. 

The  sublimation  test  may  also  be  extended  to  other  crystalline 
substances  which  undergo  sublimation  on  exposure  to  heat. 

3.  Curcuma  Thread  Test  for  Boric  Acid. — Boil  5  grams  of 
powdered  curcuma  in  10  cc.  of  alcohol.  To  the  evaporated  alco- 
hohc  extract  add  a  little  soda  and  several  cc.  of  50  per  cent, 
alcohol.  In  this  place  paper  (bast  fiber),  cotton  or  linen  threads 
and  bring  to  a  brisk  boil  for  a  few  moments.  Remove  threads 
and  dry  between  blotting  paper,  lay  them  in  a  very  weak  solu- 
tion of  sulphuric  acid  and  rinse  in  water.  When  dry  the  threads 
should  be  a  golden  yellow. 

1  Alcohol  lamp  is  preferable  because  the  flame  is  small  and  yet  the  heating  is  more 
quickly  done. 


12  MICRO-ANALYTICAL   METHODS 

The  test  for  the  presence  of  boric  acid  (also  for  borax)  is  made 
as  follows:  Dip  the  end  of  a  prepared  thread  in  a  lo  per  cent, 
solution  of  hydrochloric  acid  and  allow  to  dry.  Lay  the  thread  on 
a  slide,  cover  with  cover  glass  and  examine.  It  should  be  of  a 
reddish-brown  color.  To  the  edge  of  cover  glass  apply  a  droplet 
of  a  lo  to  13  per  cent,  solution  of  sodium  carbonate,  followed 
by  a  droplet  of  the  suspected  solution.  In  the  presence  of  boric 
acid,  the  thread  is  colored  blue,  which  coloration  remains  for  a 
longer  or  shorter  period  and  then  changes  to  gray  and  violet.  The 
test  is  a  very  delicate  one  and  is  not  hindered  by  the  presence  of 
sodium  chloride,  magnesium  sulphate  and  aluminium  sulphate. 
Strong  solutions  of  phosphoric  acid,  silicic  acid,  calcium  chlorite 
and  magnesium  chlorite,  interfere  with  the  reaction  more  or  less. 

4.  Formaldehyde  Test. — Concentrated  hydrochloric  acid 
added  to  weak  solutions  of  formaldehyde  (i  :  5000)  or  substances 
containing  formaldehyde,  forms  stellate  clusters  having  a  some- 
what crystalline  appearance.  The  formaldehyde  can  be  de- 
posited on  a  slide  by  sublimation  (as  for  benzoic  acid)  and  the 
acid  added.  The  stellate  clusters  appear  upon  evaporation 
of  the  hydrochloric  acid.  The  test  requires  further  verification 
to  determine  its  value. 

5.  Sulphurous  Acid  Test. — Moisten  starch  paper  with  a  very 
dilute  solution  of  potassium-iodide  iodine  solution  which  colors 
it  blue.  In  the  presence  of  the  merest  trace  of  sulphurous  acid 
the  paper  is  decolorized.     Do  not  use  heat  in  this  test. 

6.  Iodine  Reaction. — The  color  reaction  of  starch  with  N/50 
iodine  solution  is  of  great  importance  in  the  examination  of  fruit 
products,  such  as  jams,  jellies,  catsups,  etc.,  as  it  shows  whether 
or  not  ripe  or  green  fruits  and  juices  of  unripe  fruit  were  used 
and  whether  or  not  starch  paste  may  have  been  added  as  a  filler 
or  thickening  agent.  As  is  known,  green  fruits  generally  contain 
more  or  less  starch,  whereas  ripe  fruits  are  quite  generally  free 
from  starch.  The  reaction  may  be  observed  only  in  the  fruit 
pulp  cells,  indicating  the  presence  of  unripe  fruit,  or  it  may  be 


SPECIAL   TESTS  1 3 

limited  to  the  non-cellular  portions  of  such  substances  as  jams  and 
jellies,  indicating  the  use  of  fruit  juices  obtained  from  unripe 
fruits. 

7.  Microscopical  Examination  of  Bacteria  and  Metals  by 
Direct  Sunlight.^ — Very  minute  quantities  of  certain  minerals 
as  iron,  copper,  mercury,  and  a  few  others,  can  be  detected  in 
liquids  and  semiliquids  (in  the  form  of  metallic  hydroxides) 
when  examined  (on  slide  mounts)  by  means  of  direct  sunlight. 
All  transmissible  light  must  be  cut  off. 

Direct  sunlight  can  also  be  used  in  making  bacterial  counts  in 
liquids,  using  the  Thoma-Zeiss  hemacytometer  (Turck  ruling). 
The  bacteria  are  readily  recognizable  on  the  dark  background, 
standing  out  far  more  clearly  than  in  the  usual  examination  by 
transmitted  light,  because  of  the  more  pronounced  color  contrasts . 

8.  Micro-gluten  Test. — Mount  a  bit  of  the  flour  in  water  on  a 
slide,  beii^g  careful  not  to  use  too  much  water.  Cover  with 
cover  glass  and  move  cover  glass  to  and  fro  a  few  times  on  the 
mounted  material.  The  gluten  separates  into  stringy  fragments 
which  may  readily  be  seen  under  the  low  power  of  the  compound 
microscope.  The  use  of  a  weak  solution  of  carbol-fuchsin, 
sofranin,  or  other  stain,  will  bring  out  the  gluten  particles  more 
clearly. 

9.  Hand  Gluten  Test. — Moisten  wheat  flour  with  water,  making 
it  into  a  dough.     Knead  constantly  and  carefully  under  a  slow 

^  The  optical  principles  of  the  ultra-microscope  of  Zsygmondy  and  Siedentopf 
depend  upon  the  use  of  direct  sunlight  (or  other  intense  light)  combined  with  an 
absolutely  dark  field,  with  or  without  the  use  of  a  condenser,  the  rays  of  light  being 
directed  upon  the  object  to  be  examined  approximately  at  right  angles  to  the 
optical  axis  of  the  compound  microscope. 

The  limits  of  vision  with  the  ultra-microscope  are  approximately  0.003/x,  however, 
soUd  particles  (as  of  metallic  colloids)  of  not  more  than  0.003^1  in  diameter  show  no 
structure,  they  appear  rather  as  points  of  light. 

The  limits  of  vision  with  the  ordinary  microscope  are,  for  air  (white  light)  about 
0.30^1,  for  homogeneous  immersion  (white  light)  about  0.25/1*,  and  for  homogeneous 
immersion  when  rays  of  shorter  wave  length  than  white  light  (as  the  blue  spectrum) 
are  used,  are  about  0.15/t. 


14  MICRO-ANALYTICAL  METHODS 

stream  of  water,  washing  out  all  of  the  starch.  The  gluten  sepa- 
rates out  as  a  tenacious  gummy  mass.  With  care  fairly  accurate 
quantitative  results  may  be  obtained.  Weigh  the  dried  flour 
and  compare  with  weight  of  the  dried  gluten  mass.  With  cereal 
flours  other  than  wheat,  the  entire  dough  mass  is  gradually  washed 
away,  leaving  no  gluten. 

10.  Agar  in  Jams,  Jellies  and  Similar  Fruit  Products. — The 
method  generally  recommended  is  to  ash  a  sample  of  the  jam  or 
jelly  at  as  low  a  temperature  as  possible,  and  to  add  weak  hydro- 
chloric acid  for  the  purpose  of  decomposing  the  carbonates,  etc. 
If  agar  has  been  added  to  the  substance  the  silicious  skeletons 
of  diatoms  will  appear  in  the  ash  residue  examined  under  a  com- 
pound microscope. 

A  far  better  method  is  to  dissolve  (with  heat)  about  lo  grams 
of  the  substance  in  200  cc.  of  distilled  water  and  centrifugaHze 
(while  still  hot)  for  half  an  hour.  Decant  off  the  supernatant 
liquid  and  examine  the  residue  microscopically.  If  agar  has  been 
added,  characteristic  agar  diatoms  (mostly  Arachnodiscus  ehren- 
hergii  Baillon)  will  be  found,  also  undissolved  agar  cell  fragments 
and  remnants  of  undissolved  parasitic  algal  forms,  which  are 
quite  universally  found  upon  agar.  The  undissolved  agar  rem- 
nants and  the  algal  parasites,  which  are  in  fact  almost  as  character- 
istic as  the  diatoms,  would  be  wholly  destroyed  by  the  ashing 
process.  Furthermore,  the  ashing-acid  process,  no  matter  how 
carefully  done,  results  in  a  comminution  and  destruction  of  some 
of  the  diatom  shells.  Finding  one  or  more  diatoms  and  one  or 
more  algal  remnants  in  one  slide  mount  (or  in  5  to  20  fields  of 
view)  is  conclusive  evidence  that  agar  has  been  added,  though 
this  does  not  indicate  the  exact  amount  that  is  present.  If  the 
characteristic  structures  (diatoms  and  algal  remnants)  are  com- 
paratively abundant  then  it  is  safe  to  conclude  that  agar  has  been 
added  in  considerable  amount  (2-4  per  cent.)  or  that  an  impure 
grade  of  agar  was  used.  The  purer  the  grade  of  agar  the  fewer 
are  the  diatoms  present,  but  no  agar  has  yet  been  found  on  the 


SPECIAL  TESTS  1 5 

market  which  is  wholly  free  from  diatoms,  undissolved  agar  cells 
and  algal  parasites. 

The  reason  why  distilled  water  should  be  used  in  making  the 
solution  for  centrifugalizing  is  because  ordinary  hydrant  water 
may  contain  diatoms,  which  might  be  confusing,  especially  to  a 
beginner,  although  the  marine  diatoms  are  mostly  quite  different 
in  form  from  the  fresh  water  diatoms.  With  a  high-speed  centri- 
fuge less  material  and  less  time  need  be  consumed.  Also,  the 
more  complete  the  solution  the  better  the  results. 

8.  Micro -chemical  Color  Reaction  Tests 

There  are  certain  micro-chemical  color  reactions,  other  than 
those  already  ^mentioned,  which  are  of  great  value  in  determining 
the  presence  of  impurities  or  adulterants  in  liquids  and  semi- 
liquids.  The  methods  as  perfected  by  F.  Emich  depend  upon  the 
use  of  cotton  fibers  treated  with  certain  chemicals  which  convert 
the  metallic  compounds  into  the  sulphides.  The  prepared  threads 
can  be  readily  transferred  to  the  several  solutions  used  and  the 
color  and  precipitation  effects  can  be  observed  under  the  micro- 
scope. The  following  are  the  more  important  reagents  and 
reactions : 

1.  Cotton  Threads  for  Metal  Tests. — Dip  absorbent  cotton  threads  alternately 
into  15  per  cent,  solutions  of  sodium  sulphide  and  zinc  sulphate,  pressing  between 
blotting  paper,  and  air-dry  each  time. 

The  threads  thus  prepared  should  assume  a  deep  black  color  with  a  i  per  cent, 
solution  of  silver  nitrate.  They  may  be  kept  for  a  long  time  and  are  used  to  demon- 
strate the  presence  of  As,  Sb,  Au,  Pt,  Cu,  Hg,  Pb  and  Bi,  in  various  chemical 
compounds. 

2.  Ammonium  Sulphide  Vapor  Test. — Place  a  few  fibers  of  absorbent  cotton  into 
a  drop  of  the  suspected  solution  and  allow  the  moisture  to  evaporate.  Suspending 
the  threads  in  the  vapor  of  ammonium  sulphide  will  indicate  the  presence  of  Cd, 
Hg,  Ag,  Fe,  Co  and  Ni  (dark  to  black  coloration). 

The  prepared  threads  are  used  in  the  following  tests: 

a.  Arsenical  Test. — Dip  a  sodium  sulphide  thread  into  the  suspected  solution 
and  allow  to  dry.  In  the  presence  of  0.008  per  cent,  arsenic  there  is  a  distinct  yel- 
lowish coloration,  due  to  the  sulphide  of  arsenic  formed  in  and  upon  the  threads. 
The  arsenical  threads  will  also  show  the  characteristic  reactions  with  hydrochloric 


1 6  MICRO- ANALYTICAL   METHODS 

acid,  ammonia  and  ammonium  sulphide  by  bringing  a  drop  of  the  reagent  in  contact 
with  the  thread  upon  the  slide.     (See  also  Biological  Test  for  Arsenic  in  Part  II.) 

b.  Zinc  Test. — Dip  cotton  fibers  into  the  suspected  solution,  allow  the  moisture 
to  evaporate,  and  then  dip  the  threads  into  a  solution  of  gold  chloride.  A  violet 
coloration  develops  which  remains  in  the  presence  of  acids  but  vanishes  in  the  presence 
of  chlorine  water,  indicating  the  presence  of  zinc  chlorite.  The  reaction  is  appreciable 
in  the  presence  of  0.003  Mg  of  zinc  chlorite,  whereas  in  the  form  of  the  sulphite, 
0.1  ng  of  zinc  is  required  to  show  the  reaction. 

c.  Antimony  Test. — Dip  a  sulphide  thread  into  the  solution,  allow  solution  to 
evaporate  and  then  expose  the  thread  to  the  vapor  of  ammonium  sulphide.  If 
the  solution  to  be  tested  contains  considerable  hydrochloric  acid,  sulphide  of  anti- 
mony is  formed  upon  evaporation.    . 

d.  Gold  Test. — Gives  a  brown  coloration  with  the  sulphide  thread,  which  color 
disappears  upon  prolonged  exposure  to  ammonium  sulphide,  more  quickly  on  ex- 
posure to  chlorine,  bromine  and  sodium  hypochlorite.  The  threads  which  have 
been  decolorized  with  chlorine  are  colored  blue  to  black  with  iron  chlorite  and  violet 
to  red  with  zinc  chlorite. 

e.  Silver  Test. — A  neutral  or  faintly  acid  silver  nitrate  solution  gives  a  brown  to 
black  coloration  with  the  sulphide  thread,  the  depth  of  the  reaction  depending  upon 
the  concentration  of  the  solution.  The  fibers  can  be  decolorized  by  placing  in  sodium 
hypochlorite,  and  the  color  can  be  restored  by  means  of  zinc  chlorite  or  an  alkaline 
solution  of  grape  sugar.     Sulphuric  acid  will  again  decolorize. 

/.  Mercuric  Chloride. — Cotton  threads  dipped  into  a  solution  containing  mer- 
curic chloride  and  exposed  to  the  vapors  of  ammonium  sulphide  or  ammonia,  are 
colored  black.  The  color  is  quite  permanent  in  the  presence  of  acids.  A  sulphide 
thread  is  colored  yellow  in  neutral  solution  of  mercuric  chloride,  changing  to  black 
in  the  ammonium  sulphide  vapor. 

g.  Lead  Test.^ — Neutral  lead  solutions  (lead  nitrate)  turn  the  sulphide  threads 
yellow  and  black  on  prolonged  exposure  to  ammonium  sulphide.  In  acid  solutions 
the  color  reaction  with  the  sulphide  thread  is  black.  The  yellow  coloration  is 
promptly  changed  to  black  upon  exposure  to  ammonium  sulphide,  or  when  placed 
in  weak  sulphuric  acid  (i  :  15).  The  latter  reaction  distinguishes  between  lead  and 
mercury,  as  the  yellow  coloration  of  the  mercury  is  changed  very  slowly  with  dilute 
sulphuric  acid. 

h.  Bismuth  Test. — Solutions  color  the  sulphide  thread  reddish-brown.  Bromine 
causes  the  color  to  disappear.  Potassium  dichromate  causes  a  yellow  coloration, 
while  alkaline  solutions  of  zinc  chlorite  produce  a  black  coloration.  Lead  solutions 
are  not  reduced  by  alkaline  solutions  of  zinc  chlorite. 

i.  Iron  Test. — Ammonium  sulphide  vapor  gives  a  black  precipitate  which  is 
soluble  in  weak  solutions  of  hydrochloric  acid.  Potassium  ferrocyanide  gives  a 
blue  coloration. 

j.  Copper  Test. — Solutions  of  copper  sulphate  give  a  brown  coloration  to  the 
sulphide  thread,  which  color  persists  in  10  per  cent,  hydrochloric  acid,  but  disappears 
on  exposure  to  bromine  vapor.     The  threads  which  have  been  bleached  with  bro- 


SPECIAL   TESTS 


17 


mine  give  the  copper  ferrocyanide  reaction  when  placed  in  an  acidulated  solution  of 
potassium  ferrocyanide. 

The  following  table  from  the  work  by  Koenig  gives  the  relative 
sensitiveness  of  the  tests  above  described:^ 


Elements  in 

combination 

valency 


Reaction 


Limit 
(mg.  X  io«) 


Comparative 
sensitiveness 


Bo'" I  Curcuma  thread 

As'" i  Sulphide  thread 

Sb"' !  Sulphide  thread 

Sn" I  Violet  color  with  sulphide  thread 

Au"' I  Sulphide  thread — brown,  purple. . 

Pt"" I  Sulphide  thread 

Cu" Sulphide  thread  +  ferrocyanides. 

Ag' Sulphide  thread  +  Ag 

Hg' NH3  vapor 

Hg" Sulphide  thread 

Pb" I  Sulphide  +  PbCr04 

Bi"' !  Sulphide  +  chromate  +  Bi 

Cd" I  (NH3SH)  vapor 

Fe" ;  (NH3SH)  —  blue 

Co" i  NH3SH  or   nitroso — beta — naph- 

I  thol 

Ni" ,  NH3SH 


0.1 

m 

33,000 

lO.O 

m 

2,500 

I.O 

m 

40,000 

3-0 

in 

20,000 

3-0 

m 

22,000 

8.0 

m 

6,000 

8.0 

in 

4,000 

S-o 

in 

22,000 

8.0 

m 

25,000 

5-0 

m 

20,000 

8.0 

in 

13,000 

8.0 

m 

9,000 

6.0 

in 

9,000 

8.0 

in 

3,500 

0.3 

in 

100,000 

0-3 

in 

100,000 

9.  Making  Analytical  Reports 

The  methods  of  micro-analysts,  whether  in  private,  commercial 
or  government  laboratories,  should  be  uniform.     Much  could  be 

^  The  comparative  degree  of  sensitiveness  of  the  different  chemical  compounds 
concerned  in  the  color  reactions  above  described  and  tabulated  is  indicated  by  the 
number  of  cubic  centimeters  in  which  i  gram  of  the  substance  in  solution  is 
still  appreciable.  The  actual  limit,  determined  experimentally,  is  indicated  in 
terms  of  milligrams,  that  is  o.ooi  mg.,  represented  by  ng.  Expressing  the  com- 
parative sensitiveness  (CS)  in  a  formula  we  have 

_       Mg  limit  molecular  weights 

~  amount  limit      combination  valency 
or  to  give  the  example  for  boron,  we  have 
o.ooooi 


CS  = 


V,  59 

/:  X  =  33,000. 

0.00000006  3  ^"^' 


1 8  MICRO-ANALYTICAL   METHODS 

done  to  bring  this  about  if  the  analysts  were  to  meet  for  the 
purpose  of  comparing  methods  and  results.  Uniform  blank  re- 
port forms  should  be  adopted  and  used  in  the  micro-analytical 
laboratories,  somewhat  like  those  used  by  chemists.  It  cannot, 
however,  be  denied  that  the  efficiency  in  the  work  done  depends 
largely  upon  the  ability,  judgment  and  experience  of  the  analyst. 
The  reports  of  the  micro-analysts  may  be  made  according 
to  the  following  groups: 

I.  Drugs  and  foods  of  vegetable  origin,  including  dry  or  solid  products  of  both 
animal  and  vegetable  origin. 

II.  Liquid  or  moist  products  of  animal  and  vegetable  origin  (canned  and  pre- 
served products  generally). 

III.  Bacterial  examinations  of  liquids,  foods  and  drugs. 

There  should  be  a  special  blank  report  card  for  each  group  of 
substances,  arranged  as  follows: 

Form  No.  I 

No (I.  S.,  laboratory  or  other  serial  number). 

Label 

Sample  received Sample  examined 

Condition  of  wrappings  and  seals 

Organoleptic  tests 

Consistency  of  feel 

Color 

Odor 

Taste 

Adjunct  tests 

Sand  (beaker  test) Per  cent. 

Ash Per  cent. 

Acid-insoluble  ash Per  cent. 

Special  tests 


Microscopical  findings. 


Conclusions. 


.  Analyst. 


ANALYTICAL  REPORTS  1 9 


Form  No.  II 

(No.,  label,  dates,  condition  of  seal  and  organoleptic  tests,  as  for  form  I.) 
Adjunct  tests. 

Sublimation  tests  for 

Benzoic  acid 

Salicylic  acid 

Boric  acid  (curcuma  thread) 

Iodine  reaction. 

Intracellular 

Extracellular 

Special  tests 

Microscopical  findings. 

General 


Cytometric  counts. 

Dead  yeast  cells per  cc. 

Living  yeast  cells per  cc. 

Bacteria per  cc. 

Mold  (hyphal  fragments  and  hyphal  clusters) .  .  .  per  cc. 

Mold  spores per  cc. 

Conclusions 


,  Analyst. 


20  MICE O- ANALYTICAL  METHODS 


Form  No.  Ill 
Bacteriological  Examination 

(No.,  label,  dates,  condition  of  seals  as  for  form  I.) 

I.  Direct  count.  (Thoma-Zeiss  hemacytometer  with  Turck  ruling.) 

1.  Bacilli  per  cc 

2.  Cocci  per  cc 

II.  Plate  and  tube  cultures.     (Lactose-litmus-agar.) 

1.  Temperature  differential  test. 

a.  (20°  C.)  colonies  per  cc 

b.  is^°  C.)  colonies  per  cc 

2.  Color  differential  test. 

a.  Pink  or  yellow  colonies  per  cc 

b.  Not  pink  or  yellow  colonies  per  cc 

3.  Gelatin  liquefying  colonies  per  cc 

4 .  Indol  reaction  ( ± ) 

5.  Neutral  red  reduction  (±) 

6.  Gas  (hydrogen)  formula 

7.  Gram  stain  behavior  (±) 

8.  Presumptive  colon  bacillus  test  (±). 

a.  Amounts  used 

b.  Number  of  tests 

c.  Rating 

III.  Special  tests 

IV.  Conclusions 


Analyst. 


ANALYTICAL   REPORTS  21 

We  may  give  an  example  of  a  report  as  follows: 

Form  No.  II 

Lab.  No.  462. 

Label:    Pure  currant  jelly.     Made  by  Smith,  Jones  dr  Co.,  Nan- 
tucket, Wis. 
Sample  received  August  5,  1914.     Sample  examined  August  5, 

1914. 
Condition  of  seals:    Good,  unbroken  sample. 
Organoleptic  tests:     Not  conclusive. 

Consistency  or  feel:    Poorly  jellied. 
Color:     Normal  for  eurr ant  jelly. 
Odor :    Faint,  somewhat  disagreeable. 
Taste:     Not  characteristic,  bitterish,  quite  acid. 
Adjunct  tests. 
Sublimation  tests  for 

Benzoic  acid:     Negative. _ 
Salicylic  acid:     Very  marked. 
Boric  acid  (curcuma  thread) :     Negative. 
Iodine  reaction:     Very  marked. 
Intracellular:     Negative. 
Extracellular:     Positive,  very  marked. 
Special   tests:     Salicylic  acid  color  reaction,  with  ferric  chloride, 

very  marked. 
Microscopical  examination. 

General.     Some  apple  tissue  {udindow  cells  and  pulp  cells)  and 
currant    tissue    {selerenchyma)    present.     Added    wheat 
starch  about  $  per  cent. 
Cytometric  counts. 

Dead  yeast  cells,  80,000,000..  . per  cc. 

Living  yeast  cells,  none per  cc. 

Bacteria,  600,000,000 per  cc. 

Mold  (hyphal  fragments  and  clusters),  84,000      per  cc. 

Mold  spores,  5,000,000 per  cc. 

Smut  spores,  none per  cc. 

Conclusions:  Misbranded.  Adulterated  with  apple  and  with  wheat 
starch  and  made  from  fermented  and  decomposed  ma- 
terial, preserved  with  salicylic  acid.  Not  fit  for  human  con- 
sumption because  of  the  quantity  of  yeast,  mold  and  bacteria 
present. 

John  Doe,  Analyst. 


2  2  MICRO-ANALYTICAL  METHODS 

The  great  advantage  of  the  micro-analytical  work  as  compared 
with  chemical  work  lies  in  the  fact  that  small  amounts  of  the 
substances  are  used  for  analysis,  the  equipment  is  comparatively 
inexpensive  and  the  results  are  quickly  attained.  From  twenty 
to  forty  and  even  sixty  samples  of  simple  spices  can  be  examined 
in  one  day,  from  five  to  twelve  samples  of  powdered  vegetable 
drugs,  cocoas,  chocolates,  flours,  meals,  etc.,  and  perhaps  an 
equal  number  of  jams,  jellies,  etc. 

Because  of  the  very  close  relationship  between  the  micro- 
scopical and  bacteriological  work,  as  already  explained,  certain 
essentially  micro-analytical  methods  will  be  given  under  bac- 
teriological methods,  more  especially  in  Chapter  2  of  Part  II 
which  deals  with  the  direct  bacterial  counts,  and  also  under  milk 
analysis,  water  analysis  and  meat  analysis. 


DESCRIPTION   OF  PLATE  I 

Fig.  I. — Types  of  Pollen  Grains. — i.  Saflfron  flower.  2.  Flax.  3.  Pink.  4. 
Pumpkin  and  squash.  5.  Cloves.  Mature  pollen  grain.  6.  Cloves.  Immature 
pollen  grain.  7.  Onagraceae.  Circea  lutitiana  (Enchanter's  Nightshade).  8. 
Scutellaria.  9.  Mallow.  Distended  by  moisture.  10.  Mallow.  Normal  form. 
II.  Albuco.  12.  Lobelia  inflata.  13.  Compositse,  showing  one  mature  and  two 
immature  pollen  grains.  14.  Hibiscus.  15.  Pine  pollen.  16.  Santonica.  17. 
Mentha  species.     18.  Hyoscyamus  niger. 

Fig.  2. — ^Potato  Starch. — The  granules  are  large  and  the  markings  (hili,  lamel- 
lations)  are  distinct.  The  cross  bands  under  the  polarizer  are  very  distinct.  Potato 
starch,  mounted  in  water,  makes  a  good  test  object  for  judging  the  resolving  power 
of  objectives.  Dried  and  ground  potatoes  and  potato  parings  are  sometimes  used 
for  adulterating  purposes. 

Fig.  3. — Starches. — i.  Sago  starch  from  Cycas  revoluta  (Cycadaceae).  The 
commercial  article  known  as  sago  is  usually  in  the  form  of  small  granules  (pearl 
sago).  There  are  many  false  sagos  made  from  other  than  Cycad  or  Palm  starch. 
Much  of  this  false  sago  is  made  from  corn  starch. 

2.  Canna  starch  from  several  species  of  Canna.  The  markings  are  very  distinct, 
the  hili  being  at  the  larger  end  as  a  rule.  Also  called  arrowroot  (tous  le  mois  arrow- 
root). 

3.  Cassava  or  tapioca  starch  from  the  tuberous  roots  of  Manihot  utillissima  and 
other  species  of  Manihot.  Simple  and  compound  granules;  the  granules  are  largely 
separated  in  the  processing,  thus  giving  the  appearance  of  simple  granules.  Their 
compound  origin  is,  however,  recognizable  by  the  contact  facets. 

4.  Maranta  starch  (Arrowroot  starch)  from  Maranta  arundinacece  (Marantaceae). 
The  granules  have  many  of  the  structural  characteristics  of  potato  starch. 

5.  Yam  starch  from  several  species  of  Dioscorea  (Dioscoreaceae). 

Fig.  4. — ^Dextrinized  Starch. — The  process  of  baking  and  cooking  causes  the 
starch  granules  to  undergo  marked  structural  changes.  They  become  much 
enlarged,  the  outline  becomes  quite  indistinct  and  the  hili  and  lamellations  are 
distorted  and  correspondingly  indistinct,  i.  Normal  wheat  starch  granules.  2. 
Normal  rye  starch  granules.  3.  Dextrinized  wheat  and  rye  granules.  4.  Normal 
and  dextrinized  corn  starch.  5.  Normal  and  dextrinized  bean  starch.  6.  Normal 
and  dextrinized  ginger  starch. 


Plate  I 


Fig.  I, 


Fig.  2. 


2\fi 


m  0- 


^^^ 


Fig.  3. 


fo-o. 


t:7 


2 

) 


^J''?*^" 


(3    ^ 


f^^  A^ 


Fig.  4. 


DESCRIPTION   OF  PLATE  II 

Fig.  5. — Types  of  Crystals  of  Calcium  Occurring  in  Different  Plants. — i.  A 
parenchyma  cell  containing  a  bundle  of  needle  shaped  (acicular)  crystals  of  calcium 
oxalate  (raphide).  2,  3,  4,  Acicular  crystals  differing  in  length,  as  they  occur  in 
Scilla  and  in  other  representatives  of  the  liliaceous  groups  of  plants.  5.  Much 
elongated  prismatic  crystals  as  they  occur  in  Quillaja  and  in  Iris  florentina.  6. 
Prismatic  crystals  very  widely  distributed  in  the  plant  kingdom.  7.  Elongated 
prismatic  crystals.  8.  Twin  crystals  as  they  occur  in  Ulmus  bark.  9.  Very  large 
aggregate  crystals  as  they  occur  in  Rheum  and  Polygonum  species.  10,  11.  Smaller 
aggregate  crystals  very  widely  distributed  in  the  vegetable  kingdom.  12,  13.  Very 
minute  prismatic  (pyramidal)  crystals  as  they  occur  in  Belladonna.  14.  Prismatic 
crystals  as  they  occur  in  Hyoscyamus  and  in  other  plant  groups. 

Calcium  oxalate  crystals  are  among  the  highly  diagnostic  structural  characteris- 
tics of  drug  plants  and  should  be  studied  not  only  as  to  form  but  also  as  to  size. 
They  are  not  dissolved  in  the  usual  mounting  media  and  are  not  destroyed  by  heat. 
They  dissolve  slowly  in  the  stronger  acids  (hydrochloric  acid). 

Fig.  6. — Types  of  Bast  Cells  as  They  Occur  in  Barks  and  in  Other  Plant 
Parts. — I.  Shorter  bast  cell  as  they  occur  in  the  cinnamon  barks.  2.  Typical 
bast  cell  (showing  a  portion  of  a  cell  only)  as  they  occur  in  willaw  bark,  in  Ulmus,  in 
Mezereon,  etc.  3.  Branching  bast  cells  as  they  occur  in  Quillaja  and  in  Prunus 
bark.  4.  Greatly  thickened  sclerenchymatous  bast  cells  as  they  occur  in  the 
Cinchonas. 

Fig.  y. — Types  of  Sclerenchyma  (Stone)  Cells. — i.  Typical  sclerenchyma  cells 
as  they  occur  in  the  endocarp  of  drupaceous  fruits  and  nuts.  2.  Elongated  bast- 
like sclerenchyma  cells.  3.  Thin- walled  typical  sclerenchyma  cell.  4.  Scleren- 
chyma cell  with  unequally  thickened  walls  as  they  occur  in  the  cinnamons.  5. 
Large  thin-walled  sclerenchyma  cells  as  they  occur  in  the  seed  coat  of  Amygdala. 

6.  Branching  sclerenchyma  cells  as  they  occur  in  tea  leaves  and  in  peanut  exocarp. 

7,  8,  9.  Forms  of  sclerenchyma  cells. 

Fig.  8. — Typical  Sclerenchjmia  Cells  (in  groups)  as  they  occur  in  the  pulp  of 
the  pear. 


Plate  II 


Ftg.  5. 


f'iG.    6. 


Fig.  7. 


Fig.  8. 


DESCRIPTION   OF  PLATE  III 

Fig.  9. — ^Buckwheat. — i.  Proteid-bearing  tissue.  2.  Starch-bearing  endosperm 
tissue.  Cell  walls  are  very  thin  and  the  entire  cell  lumen  is  packed  with  starch 
granules.  3.  Starch  granules.  The  granules  resemble  those  of  corn,  being  some- 
what smaller.     4.  Sclerenchymatous  fibers. 

Buckwheat  is  the  predominating  ingredient  of  the  buckwheat  pancake  flours 
and  is  occasionally  used  as  an  adulterant  of  spices. 

Fig.  id. — Tissues  of  the  Pine. — i.  The  characteristic  tracheids  with  bordered 
pits.  2.  Bast-like  fibers  of  the  bark.  3.  Crystal-bearing  bark  parenchyma  cells. 
4.  Tracheids  in  radial  view.  5.  Medullary  ray  cells  in  radial  view.  Pine  wood 
(pulp)  is  much  used  in  making  paper. 

Fig.  II. — Sclerenchyma  Cells  of  Olive  Pits.- — Ground  olive  pits  were,  until 
recently,  extensively  employed  as  an  adulterant  of  spices  and  drugs. 

Fig.  12. — Clove  Stems. — A  very  common  adulterant  of  cloves  and  of  allspice. 
I.  Typical  sclerenchyma  cells.  2.  Sclerenchyma  cells  with  unequally  thickened 
walls.     3.  Sclerenchymatous  bast  fibers. 


Plate  III 


Fig.  9. 


Fig.  to. 


Fig.  II. 


Fig.  12. 


DESCRIPTION   OF  PLATE  IV 

Fig.  13. — Cassia  Buds  and  Cassia  Stems. — i.  Sclerenchymatous  fibers  of  the 
cassia  stems.  2.  Bast  fibers  of  cassia  stems.  Parenchymatous  cells  of  the  buds.  4. 
Trichomes  of  buds.  5.  Thick-walled  parenchyma  cells.  Cassia  buds  and  cassia 
stems  are  frequently  used  in  adulterating  cloves,  allspice  and  cinnamon. 

Fig.  14. — Coffee  Adulterants. — i.  Sclerenchyma  cells  of  date  pits.  2.  Scleren- 
chyma  cells  of  the  walnut  shell.  3,  4,  5.  Tracheids  and  inulin-bearing  parenchyma 
cells  of  chicory.  Figs  and  prunes  are  also  much  used  as  coffee  adulterants,  also 
cereals,  fleshy  roots,  acorns,  etc. 

Fig.  15. — ^Wheat  Tissues. — i.  Wheat  starch.  2.  Trichomes  from  the  bran. 
3.  Starch-bearing  parenchyma.  4.  Epicarp  cells.  5.  Proteid-bearing  cells  from 
middlings.  Rye  histology  is  similar  to  that  of  wheat.  Wheat  flour  is  used  in 
macaroni,  spaghetti,  noodles,  etc.  Wheat  flour,  bran  and  middlings  are  much 
used  for  adulterating  purposes.  Rye  starch  differs  from  that  of  wheat  in  the  larger 
size  of  the  granules  and  the  greater  prominence  of  the  hili. 

Fig.  16. — ^Rice  Tissues. — i.  Starch.  Single  granules  and  aggregates.  These 
aggregates  are  characteristic  of  rice  and  of  oats.  2.  Starch-bearing  endosperm 
cells.  3,  4,  5.  Epicarp  and  pericarp  cells.  In  form  the  starch  granules  of  rice,  oat, 
corn,  darnel,  millet,  fox-tail,  buckwheat  and  chess  resemble  each  other.  The  size 
varies  very  much. 


Plate  IV 


h 


h 


Fig.  13. 


Fig.  14. 


,oOoo 


Fig.  15. 


Fig.  16. 


DESCRIPTION  OF  PLATE  V 

Fig.  17. — 'Bean  Tissues. — i.  Epidermal  palisade  tissue  with  the  crystal-bearing 
hypoderm.  2.  Starch-bearing  endosperm  tissue.  3.  Starch  granules  with  promi- 
nent fissured  hili.  4.  Spongy  tissue,  5.  Epidermal  palisade  cells  in  vertical  view. 
6.  Prismatic  crystals  of  calcium  from  hypoderm. 

Ground  beans,  peas  and  lentils  are  used  for  adulterating  purposes. 

Fig.  18. — ^Histology  of  Mallow  Leaf. — i.  Transverse  section  of  leaf  showing 
stellate  trichome,  epidermal,  palisade  and  spongy  tissue  cells.  Aggregate  crystals 
of  calcium  oxalate  are  present.  2.  Stellate  or  aggregate  trichomes.  3.  Epidermal 
cells  (lower)  showing  stomata.  Mallow  leaves  are  extensively  employed  for  adul- 
terating leafy  spices  and  drugs. 

Fig.  19. — Histology  of  Corn. — i.  Corn  starch.  2.  Starch-bearing  endosperm 
of  corn  kernel.  3.  Trichomes  of  the  chaff  of  the  corn  cob.  4.  Sclerenchymatous 
cells  of  the  corn  cob.  Ground  corn  cobs  are  used  for  adulterating  purposes  and 
also  in  the  manufacture  of  artificial  maple  syrup  flavor. 

Fig.  20. — ^A  Few  Types  of  Trichomes. — i.  Branching  trichome  of  mullein.  2. 
Many-celled  simple  trichome  of  henbane  showing  wart-like  marking  on  outer  surface. 
3.  Simple  single-celled  trichome  as  of  rye  and  wheat.  4.  Glandular  trichome  with 
two  secreting  cells.  5.  Glandular  trichome  with  one  secreting  cell.  6.  Many- 
celled  glandular  trichome.  7.  Simple,  single-celled  trichome  of  Indian  hemp.  8. 
Much  elongated  and  twisted  single-celled  trichome,  as  of  sage.  9.  Sessile  glandular 
trichome  (Eriodictyon).     10.  Indian  hemp.     11.  Pyrethrum.     12.  Simple  trichome. 


Plate  V 


Fig.  17. 


Fig.  18. 


To  f  ' 


Fig.  19. 


Fig.  20. 


DESCRIPTION  OF  PLATE  VI 

Illustrating  the  Histology  of  a  Typical  Bark  Showing  all  of  the  Tissues 
Which  May  be  Found  in  a  Bark. — A,  Longitudinal  section  in  the  radial  direc- 
tion but  not  showing  the  medullary  rays.  B,  Transverse  section,  i.  Outer 
bark.  The  demarkation  between  outer  and  inner  bark  is  not  always  distinct.  2. 
Inner  bark.  3.  Beginning  of  wood  tissue,  a,  Epidermis.  Always  wanting  in  tree 
trunks  and  older  branches,  b,  Cork  tissue,  c,  Bark  parenchyma.  Cell-walls  are 
usually  not  suberized  and  the  cells  may  contain  various  inclusions  such  as  crystals 
of  calcium  oxalate,  tannin,  starch  granules  and  resin,  d,  Groups  of  sclerenchyma 
cells.  These,  when  present,  normally  predominate  in  the  outer  bark,  e,  Crystal- 
bearing  fibers  which  usually  accompany  the  bast  fibers.  /,  Bast  fibers.  These, 
when  present,  normally  predominate  in  the  inner  bark.  The  fibers  may  occur  singly 
or  in  groups,  g,  Cambium,  h,  Wood  libers,  i,  Ducts.  Usually  of  the  typically 
porous  type,     k,  Medullary  rays. 

An  excellent  typical  bark  having  all  of  the  histological  elements  indicated  in 
Plate  VI  is  Rhamnus  purshiana.  The  demarkation  between  outer  bark  and  inner 
bark  is  well  defined  in  Ulmus  and  Quillaja. 


Plate  VI 


>i 


o 


Fig.  21 


II 

BACTERIOLOGICAL  METHODS  IN  FOOD  AND 
DRUGS  LABORATORIES 


I.  Introduction 


The  study  of  the  significance  of  bacteria  in  foods  of  all  kinds  is 
one  of  the  most  important  and  interesting  of  scientific  subjects 
and  one  which  has  received  much  attention  ever  since  the  science 
of  bacteriology  has  become  more  highly  developed  as  the  result 
of  the  perfection  of  the  compound  microscope.  For  a  long 
period  of  time  the  popular  notion  has  prevailed  that  bacteria  were 
essentially  harmful  and  to  designate  any  substance  as  bacterially 
contaminated  was  to  pronounce  it  dangerous  and  to  condemn  it 
vnthout  trial.  We  now  know  that  many,  in  fact  most  bacteria, 
are  beneficent  rather  than  harmful,  and  that  many  different 
species  of  bacteria  are  concerned  in  the  preparation  of  food 
substances.  It  cannot  be  denied,  however,  that  many  kinds  of 
bacteria  as  well  as  other  organisms  are  concerned  in  the  pro- 
duction of  changes  in  food  substances  which  we  know  to  be  highly 
detrimental  to  the  well  being  of  the  human  race.  It  is  the  duty 
of  modern  sanitary  science  to  guard  against  disease  and  the 
contamination  of  food  substances  through  the  invasion  of  patho- 
genic and  otherwise  objectionable  organisms.  It  is  the  work  of 
the  food  bacteriologist  to  detect  objectionable  contaminations  in 
foods  and  to  aid  in  developing  those  processes  and  methods  of 
food  preparation  and  manufacture  which  will  prevent  the  re- 
currence of  such  contamination.  The  food  bacteriologist  will 
center  his  attention  on  the  following: 

23 


24  BACTERIOLOGICAL   METHODS 

1.  Chemical  (decomposition)  changes  in  foods  and  drugs  induced  by  the  various 
organic  infecting  agents,  as  bacteria  and  other  living  organisms,  which  render  such 
substances  unfit  for  human  use  or  which  render  them  dangerous  for  human  use. 

2.  Foods  and  drugs  as  actual  or  possible  carriers  of  infecting  agents  which  are 
or  may  be  dangerous  to  life  or  which  may  or  might  be  injurious  to  the  physical 
well-being  of  the  human  species. 

It  goes  without  saying  that  the  food  bacteriologist  must  pro- 
ceed carefully  in  order  that  there  may  be  no  hasty  decisions  re- 
sulting in  the  condemnation  of  food  products  which  are  not  in- 
jurious. There  is,  however,  little  excuse  for  hasty  or  unjustifi- 
able passing  of  judgments  as  regards  the  quality  of  food.  Bac- 
teriological and  toxicological  methods  have  been  sufficiently 
perfected  so  that  the  careful  analyst  need  not  make  unfair  or 
unwarranted  decisions.  The  men  entrusted  with  the  critical 
examination  of  foods  and  drugs  as  to  their  fitness  for  human 
use  should  be  investigators  of  authority  and  should  have  had 
wide  range  of  practical  as  well  as  laboratory  experience,  and  they 
should  furthermore  be  possessed  of  good  judgment.  While 
the  condemnation  of  food  materials  should  not  be  hasty  it  should 
on  the  other  hand  not  be  too  tardy  or  conservative.  The  prime 
object  of  the  work  by  the  food  bacteriologist  is  to  protect  the 
consumer,  not  the  dealer  or  manufacturer.  This  very  important 
point  is  most  unfortunately  not  properly  heeded  with  the  result 
that  some  of  the  work  done  in  the  administrative  laboratories  is, 
or  appears  to  be,  in  the  interests  of  the  dealer  or  manufacturer. 

A  goodly  number  of  infections  enter  the  human  system  by  way 
of  the  mouth  with  the  ingested  foods  and  drinks.  Food  substances 
form  excellent  pabula  for  the  bacteria  and  other  parasitic  agents 
which  enter  the  digestive  tract  or  which  may  already  have  entered. 
Foods  and  drinks  are  exposed  to  infection  in  a  great  variety  of 
ways.  For  purposes  of  illustration  we  may  cite  bread,  the  so- 
called  '^ staff  of  life,"  as  one  of  the  foods  which  is  liable  to  infec- 
tion. It  may  be  assumed  that  the  loaf  of  bread,  when  it  is  taken 
from  the  oven,  is  entirely  sterile  and  free  from  living  organisms 
of  all  kinds.     Just  as  soon  as  the  loaf  is  cool  enough  to  permit  it, 


INTRODUCTION  2$ 

the  promiscuous  manipulation  begins  and  is  continued  until  the 
bread  enters  the  digestive  tract  of  the  consumers.     The  loaves 
are  handled  by  the  dirty,  sweaty  and  oftentimes  diseased  hands 
of  the  baker  or  his  helper.     Basketfuls  of  uncovered  bread  are 
dragged  over  the  dirty  floors,  over  sidewalks,  and  through  the 
filthy  alleys.     The  uncovered  loaves  are  repeatedly  handled  by 
the  bakery  drivers  whose  hands  and  clothing  are  generally  very 
filthy.     The  uncovered  loaves  are  left  on  doorsteps  and  other 
exposed  places  on  the  premises  of  the  consumer.     This  much- 
handled  bread  is  finally  eaten,  crust  and  all,  without  any  attempt 
at  sterilization.     Such  bread  may  be  contaminated  with  a  great 
variety  of  disease  germs.     Infections  from  hands,  disease-bearing 
dust  from  the  streets  and  alleys,  excreta  from  disease-carrying 
flies,  excreta  from  the  intestinal  tract  of   man  and  of   animals 
are  among  the  deposits  which  have  been  found  on  the  exterior  of 
bread.     Miss  Katherine  Howell  has  traced  an  epidemic  of  typhoid 
fever  to  the  consumption  of  contaminated   bread   and   she   has 
demonstrated  the  presence  of  typhoid  fever  germs  and  of  in- 
testinal bacteria  on  numerous  loaves  of  bread.     Edward  Bartow, 
director  of  the  Illinois  State  Water  Survey,  has  also  demonstrated 
a  bread-borne  typhoid  epidemic  in  Rockford,    Illinois.     Colon 
bacilli  are  usually  found  in  considerable  numbers  on  every  loaf 
of  unwrapped  bread.     Every  loaf  of  bread  from  the  public  bakeries 
should  be  wrapped  in  sterilized  paper  bags  just  as  soon  as  it  leaves 
the  oven  and  it  should  remain  in  these  bags  until  ready  to  be 
placed  before  the  consumer. 

Polluted  water  may  carry  the  germs  of  dysentery,  of  cholera, 
of  typhoid  fever  and  the  larvae  of  intestinal  and  other  parasites. 
Clams  and  oysters  have  caused  typhoid  epidemics.  Fruits  and 
vegetables  are  frequently  polluted  with  fertilizer,  especially  where 
human  fertilizer  is  used,  as  is  the  custom  with  the  Chinese  truck 
gardeners  not  only  in  China  but  also  in  other  lands  where  the 
Chinese  are  found.  Using  human  excrement  as  a  fertilizer  of 
soil  should  be  prohibited  by  law.     American  army  surgeons  at 


26  BACTERIOLOGICAL  METHODS 

the  time  of  the  American  occupation  of  Cuba  made  the  filthy 
farming  customs  of  the  Chinese  the  object  of  a  special  report  but 
apparently  nothing  ever  came  of  the  recommendations  made. 
The  Chinese  also  import  dried  human  feces  and  dried  human 
urine  for  medicinal  purposes  and  a  recommendation  was  made 
to  Washington  to  prohibit  such  importations  but  apparently 
nothing  has  ever  been  done  about  it. 

Pollution  of  fruits  and  berries  of  all  kinds  may  come  from 
the  hands  of  pickers.  Gathering  of  fruit  is  usually  done  by  the 
very  ignorant,  those  who  have  no  proper  conception  of  personal 
cleanliness  and  of  sanitation.  Entire  families,  men,  women,  and 
children,  migrate  to  the  fields  and  work  during  the  hottest  part  of 
the  season.  They  live  in  the  open  or  in  tents  or  perhaps  in  covered 
wagons.  The  environment  of  these  temporary  abiding  places 
is  anything  but  sanitary.  Sickness  often  prevails  in  these  camps, 
such  as  typhoid  fever,  scarlet  fever,  measles  and  dysentery,  to  say 
nothing  of  the  more  common  body  and  intestinal  parasites  which 
infest  many  of  the  laborers.  These  multitudinous  infections  are 
brought  in  contact  with  the  fruit,  berries,  peas,  beans,  lettuce, 
cabbage,  cucumbers,  etc.,  etc.  The  products  of  the  field  are  then 
carried  to  the  consumer  by  a  driver  who  disseminates  the  contami- 
nation by  mixing  and  frequent  handling.  And  in  spite  of  all  this 
there  are  those  who  insist  on  eating  berries  unwashed  because 
they  might  lose  some  of  the  natural  flavor. 

Next  to  bread,  milk  is  the  most  popular  food  substance.  Most 
unfortunately  milk  is  also  one  of  the  best  food  substances  for 
all  manner  of  germs,  harmful  and  harmless.  Sickness  in  those 
employed  about  the  dairying  establishment  has  time  and  again 
caused  epidemics,  such  as  diphtheria,  typhoid  fever,  scarlet  fever, 
tuberculosis,  dysentery,  and  streptococcic  tonsillitis.  Diseased 
animals  transmit  infection  to  humans,  as  will  be  more  fully  ex- 
plained in  the  chapters  following. 

It  is  generally  believed  that  the  usual  processes  of  baking  and 
cooking  as  practised  in  the  household  are  a  sure  guarantee  that 


INTRODUCTION  27 

the  foods  so  prepared  are  entirely  free  from  living  bacterial  infection 
of  all  kinds.  This  is  true  of  some  foods  but  not  by  any  means  of 
all  of  them.  Dr.  W.  A.  Sawyer,  Director  of  the  CaHfornia  State 
Hygienic  Laboratory,  in  reporting  upon  an  epidemic  of  93  cases  of 
typhoid  fever  (at  Hanford,  California)  due  to  a  single  carrier, 
traced  the  source  of  the  infection  to  cooked  Spanish  spaghetti, 
prepared  by  the  typhoid  carrier.  The  following  tests  were  made 
in  the  California  State  Hygienic  Laboratory  to  ascertain  the  effects 
of  baking  on  the  presence  of  typhoid  fever  germs  in  the  interior  of  a 
mass  of  spaghetti. 

"A  large  hot-air  sterilizer  was  heated  and  kept  between  160° 
and  170°  C.  (320°  and  338°  F.).  The  pan  of  spaghetti  was  in- 
troduced and  subjected  to  this  heat  for  30  min.  When  the  dish 
was  removed  the  surface  was  of  a  golden  brown  color.  The  ap- 
pearance and  aroma  suggested  that  the  spaghetti  was  thoroughly 
cooked  and  very  hot.  The  temperature  near  the  top  was  54°  C. 
(129.2°  F.)  and  at  the  middle,  23°  C.  (73.4°  F.).  Ten  minutes 
later  the  temperature  at  the  middle  was  24°  C.  (75.2°  F.)  and  the 
dish  was  then  returned  to  the  oven.  Cultures  taken  at  various 
levels  showed  that  the  typhoid  bacilli  were  alive  even  close  to  the 
surface. 

"In  the  next  baking  the  oven  was  kept  at  temperatures  ranging 
between  207°  and  214°  C.  (405°  to  417°  F.).  After  half  an  hour 
the  pan  was  removed.  The  surface  was  dark  brown  and  the 
points  sticking  up  from  it  were  charred.  The  liquid  around  the 
margin  was  boiling  vigorously  and  the  whole  dish  was  sizzling. 
The  temperature  just  under  the  surface  was  83°  C.  (181.4°  F.). 
At  the  middle  it  was  28°  C.  (82.4°  F.)  and  near  the  bottom  it 
was  48°  C.  (118.4°  F.).  An  hour  later  the  temperatures  had  be- 
come nearly  equalized  and  were  46°  C.  (114.8°  F.)  near  the  top, 
42.5°  C.  (108.5°  F.)  at  the  middle,  and  43°  C.  (109.4°  F.)  near  the 
bottom.  This  showed  that  the  interior  of  the  dish  did  not  reach 
even  a  pasteurizing  temperature. 

"  Cultures  taken  at  the  surface  soon  after  the  pan  had  been 


28  BACTERIOLOGICAL   METHODS 

removed  from  the  oven  showed  no  typhoid  colonies  and  very  few 
of  other  kinds.  Cultures  taken  at  a  distance  of  half  an  inch  from 
the  surface  showed  a  few  colonies  of  the  typhoid  bacillus,  most  of 
the  organisms  having  been  killed.  Cultures  from  a  depth  of 
2^^  in.  showed  abundant  colonies  of  typhoid  bacilli.  In  these 
cultures  the  typhoid  colonies  were  identified  by  their  appearance 
on  Endo  medium  and  Russell  medium  and  also  by  agglutination 
by  anti-typhoid  serum." 

Dr.  Sawyer  sums  up  the  experimental  evidence  as  follows: 

"The  laboratory  experiments  completed  the  explanation  of  the 
Hanford  outbreak  by  showing  that  the  sauce  used  in  making  the 
Spanish  spaghetti  was  a  good  culture  medium  and  that  the  dish 
had  not  been  sterilized  after  leaving  the  house  of  the  typhoid 
carrier. 

'' Moreover,  it  was  demonstrated  that  cooked  dishes  mus1: 
be  considered  as  possible  conveyors  of  infection  unless  it  can  be 
shown  that  the  method  of  cooking  would  produce  complete 
sterilization.  The  slowness  with  which  heat  penetrates  dishes 
like  the  Spanish  spaghetti  shows  that  very  prolonged  heating 
would  be  necessary  for  sterilization  of  large  dishes  of  such  food. 
Ordinary  baking  merely  incubates  the  interior  of  these  masses 
of  food." 

This  report  by  Dr.  Sawyer  is  of  special  significance  to  the 
food  bacteriologist  as  it  illustrates  two  very  important  factors 
concerned  in  the  study  of  food  sanitation:  First,  the  possible 
contamination  of  food  materials  through  carriers  of  disease, 
and  secondly,  the  necessity  of  studying  more  carefully  our  pres- 
ent methods  of  sterilization  (of  food  materials)  through  the 
agency  of  heat.  As  will  be  more  fully  set  forth  in  subsequent 
chapters,  the  examination  of  canned  foodstuffs  shows  that  sterili- 
zation is  far  from  complete  in  the  great  majority  of  cases. 

In  addition  to  the  more  or  less  acute  infections  traceable  to 
the  consumption  of  contaminated  food  products,  there  are  the 
multitudinous    infections    which    are    of    slow    development    or 


INTRODUCTION  29 

chronic  in  character.  In  many  of  these  cases  it  is  not  possible 
to  acertain  definitely  how  the  infection  entered  the  system. 
There  are  numerous  so-called  autointoxications  which  are  said 
to  be  due  to  autolytic  changes  in  the  ingested  food  substances 
resulting  in  the  formation  of  toxins  which  often  give  rise  to  very 
serious  and  even  fatal  poisoning.  As  is  generally  known,  certain 
toxin-forming  bacteria  after  once  gaining  access  to  the  intestinal 
tract  may  remain  there  for  years  feeding  upon  the  contents  of 
the  intestines  and  producing  enough  of  the  toxin  to  give  rise  to 
symptoms  of  poisoning  of  a  more  or  less  chronic  character.  In 
some  instances  the  toxin-forming  bacteria  are  not  present  in  suf- 
ficient numbers  or  do  not  multiply  in  sufficient  numbers  to  give 
rise  to  any  marked  symptoms,  and  in  still  other  cases  the  originally 
pathogenic  or  toxin-forming  bacteria  lose  their  virulency  after 
having  lived  in  the  intestinal  tract  for  some  time.  As  is  known, 
there  is  constant  warfare  in  the  intestinal  tract  between  the  harm- 
ful and  the  really  beneficent  bacteria,  and  it  is  this  discovery  which 
has  led  Metschnikoff  and  other  bacteriologists  to  find  germs 
which  upon  being  introduced  into  the  intestinal  tract  would 
overcome  or  crowd  out  the  objectionable  toxin  formers. 

Food  poisoning  has  received  considerable  attention  in  re- 
cent years.  Vaughan  and  Novy  have  suggested  a  nomenclature 
applicable  to  certain  recognizable  forms  of  poisonings  traceable 
to  foods,  as: 

Bromatotoxismus  or  food  poisoning. 
Galactotoxismus  or  milk  poisoning, 
Tyrotoxismus  or  cheese  poisoning. 
Kreatoxismus  or  meat  poisoning. 
Ichthyotoxismus  or  fish  poisoning. 
Mytilotoxismus  or  mussel  poisoning. 
Sitotoxismus  or  cereal  poisoning. 

The  poisonings  mentioned  are  generally  due  to  toxins  or 
related  products  elaborated  by  bacteria,  but  in  some  instances  the 
exact  species  responsible  for  such  toxin  formation  have  not  yet 
been  determined.     The  identification  of  the  species  of  bacteria 


30  BACTERIOLOGICAL  METHODS 

responsible  for  the  poisoning  of  foods  and  drinks  is  of  minor  im- 
portance. What  is  of  prime  importance  to  the  food  bacteriologist 
is  to  find  the  poison  and  if  possible  to  ascertain  the  manner  in 
which  the  poison  gained  access  to  the  food  substance,  in  order  that 
methods  may  be  devised  to  guard  against  the  recurrence  of  such 
contamination.  It  may  also  be  stated  that  in  the  great  majority 
of  cases  of  food  poisoning  the  nature  of  the  poison  and  its  source 
have  already  been  determined  and  means  are  available  to  pro- 
tect the  consumer.  If  the  manufacturers  of  foods  and  of  food 
products  would  give  proper  attention  to  the  modern  methods  of 
manufacture,  then  poisonings  due  to  the  eating  of  such  prod- 
ucts will  be  a  rare  occurrence  indeed.  It  is  regrettable  that  so 
many  of  the  smaller  establishments  engaged  in  the  manufacture 
of  food  products  are  not  better  informed  regarding  the  available 
modern  methods  of  preparing  and  storing  food  substances  in  such 
a  manner  as  to  guard  against  infection  and  contamination.  It 
is  also  regrettable  that  the  various  pure  food  and  drugs  laws  and 
regulations  intended  to  protect  the  consumer  are  not  more  effi- 
ciently and  more  strictly  enforced. 

We  have  already  suggested  a  more  efficient  coordination  of 
the  chemical,  microscopical  and  bacteriological  methods  of  analy- 
sis in  our  food  and  drugs  laboratories — federal,  state,  munici- 
pal and  private.  The  following  are  the  bacteriological  methods 
applicable  in  the  examination  of  foods  and  drugs  as  to  quality 
and  purity.  It  is  hoped  that  the  suggestions  offered  may  serve 
as  a  basis  for  establishing  more  complete  practical  working 
methods  and  at  the  same  time  indicate  lines  for  further  research. 

Just  what  bacteriological  analyses  and  tests  should  be  made 
in  pure  food  and  drugs  laboratories  has  as  yet  not  been  decided 
upon.  However,  based  upon  the  present  purpose  and  scope  of 
such  laboratories,  we  submit  the  following  outline  as  covering 
the  field  fairly  well  and  which  outline  will  be  followed  quite  closely 
in  the  text,  however  not  necessarily  adhering  to  the  same  sequence 
of  the  subject-matter. 


introduction  3 1 

Quantitative  and  Qualitative  Determinations  of  Organisms 
IN  Foods  and  Drugs 

Substances  to  be  analyzed. 
Liquids  of  all  kinds. 

Semiliquids  and  semisolids  miscible  with  water. 
Solids  of  all  kinds. 
Numerical  and  quantitative  limits  of  contamination  in  different 
substances. 
For  molds — quantity  of  spores  and  hyphae. 
For  yeasts — number  and  kind. 
For  bacteria — number  and  kind. 
For  pus,  dirt,  sand,  etc. 
Methods. 

Making  concentrations. 

Making  dilutions. 

Making  the  counts  and  estimates. 

Bacteria. 

Yeasts. 

Mold  spores  and  mold  hyphae. 

Algae,  in  drinking  waters,  etc. 

Protozoa. 

Pus  cells,  in  milk,  etc. 

Dirt,  sand,  etc. 
Plate  counts — Petri  dish  cultures. 

Culture  media  used. 

Optimum  temperature. 

Time  of  incubation. 
Qualitative  determinations. 
Apparatus. 
Culture  media. 
Stains. 
Special  methods. 

Colon  group  of  bacilli. 

Presumptive  colon  bacillus  test. 

Sewage  streptococci. 

Dysentery  bacilli  and  amebae. 

Bacillus  typhosus. 

Paratyphoid  group. 

Cholera  vibrio. 

Yeasts. 

Molds. 

Animal  parasites. 

Larvae,  ovae,  etc. 


32  BACTERIOLOGICAL  METHODS 

Biological  water  analysis. 

Bacteria,  number  and  kind. 

Diatoms. 

Desmids. 

Nostoc. 

Other  algae. 

Molds;  significance  of. 
Bacteriological  milk  analysis. 

Quantitative. 

Standards  for  different  geographic  areas. 

Summer  and  winter  standards — temperature  standards. 

Qualitative. 

Pus  and  blood  corpuscles;  significance  of. 

Milk  diseases. 
Blue  milk. 
Ropy  milk. 
Bad  odors,  bad  taste,  etc. 

Sour  milk. 

''Buttermilk"  tablets. 

Kefir,  koumys,  etc. 
Bacteriological  examination  of  shellfish. 
The  bacteriological  and  toxicological  examination  of  meat 

and  meat  products. 
The  bacteriological  examination  of  eggs  and  of  egg  products. 
Bacteriological  examination  of  mineral  waters. 
Bacteriological  examination  of  pharmaceuticals. 
Bacteriological  examination  of  sera,  vaccines,  bacterins,  etc. 
The  microscopical  and  bacteriological  examination  of  syrups. 
Standardization  of  disinfectants. 

Phenol  coefficient. 

Albumen  coagulation  coefficient. 

Toxic  coefficient. 

The  efficiency  value  of  disinfectants. 
Biological  toxicity  tests. 

Upon  first  consideration  it  would  appear  that  the  bacteriolog- 
ical methods  in  food  and  drugs  laboratories  might  be  closely  simi- 
lar to  those  in  hygienic  laboratories.  Such  is  the  case  in  a  gen- 
eral way,  however,  with  certain  well-defined  differences.  Whereas 
the  bacteriological  work  in  hygienic  laboratories  pertains  to  the 
prevention  of  disease  and  finding  the  primary  causes  of  disease^ 
the  work  in  the  food  and  drugs  laboratories  has  to  do  with  the 


INTRODUCTION  33 

investigation  of  the  biological  factors  influencing  the  quality  of 
food  and  drugs  and  the  significance  of  pure  food  and  drugs  in 
the  maintenance  of  the  public  health  and  the  physical  well-being 
of  the  human  race,  as  against  the  pernicious  effects  of  contami- 
nated foods  and  drugs. 

The  question  for  first  consideration  is  what  bacteriological 
methods  are  necessary  and  practicably  applicable  in  testing 
foods  and  drugs?  This  phase  of  the  subject  is  comparatively 
new  and  accordingly  there  are  but  few  food  and  drugs  bacteri- 
ologists who  have  had  any  considerable  general  range  of  ex- 
perience, and  as  a  consequence  there  are  comparatively  few 
methods  fully  worked  out.  Most  of  the  bacteriological  in- 
vestigations and  researches  pertaining  to  foods  have  been  along 
special  lines,  and  indeed  much  valuable  information  and  useful 
data  have  been  brought  together.  Within  the  last  lo  years  the 
work  on  the  sanitary  examination  of  milk  and  of  water  supplies 
has  become  monumental  in  volume  as  well  as  in  importance. 
Numerous  methods  have  been  tried,  some  to  be  entirely  abandoned 
after  being  for  a  time  heralded  as  the  final  word  in  determining 
the  potability  of  water  supplies.  The  same  may  be  said  of  the 
development  of  the  bacteriological  examinations  of  milk  supplies. 

Quite  recently  bacteriologists  have  given  considerable  atten- 
tion to  the  sanitary  examination  of  shellfish,  more  especially 
with  reference  to  sewage  contamination.  In  this  investigation 
American  bacteriologists  have  taken  the  lead.  European  bac- 
teriologists have  done  an  enormous  amount  of  work  in  the  ex- 
amination of  sewage  and  of  sewage  disposal,  to  say  nothing  of 
the  classical  researches  on  yeasts  and  on  fermentation  in  general. 
However,  the  general  bacteriology  of  foods  and  of  drugs  is  as  yet 
an  unexploited  field.  It  is  true,  the  Bureau  of  Chemistry  of 
the  Department  of  Agriculture  has,  within  recent  years  (since 
1906),  done  considerable  work  on  the  sewage  contamination  of 
oysters  and  other  shellfish  (Bulletin  No.  136,  Bureau  of  Chemistry. 
U.  S.  Department  of  Agriculture,  by  George  W.  Stiles)  and  in 


34  '  BACTERIOLOGICAL  METHODS 

the  quantitative  estimation  of  the  microbic  contamination  of 
certain  food  supplies,  and  still  more  recently  the  laboratory 
division  of  the  U.  S.  Public  Health  Service  has  done  much  efhcient 
work  on  the  standardization  of  disinfectants.  We  must  also 
mention  the  work  on  milk,  meat  inspection,  etc.,  by  the  Bureau 
of  Animal  Industry  and  the  work  on  sanitation  and  related  sub- 
jects by  the  U.  S.  Public  Health  Service,  not  forgetting  to  men- 
tion the  vast  amount  of  routine  analyses  in  state  and  municipal 
health  laboratories  and  the  sporadic  research  work  in  the  bio- 
logical and  bacteriological  laboratories  of  our  colleges  and  uni- 
versities and  the  individual  investigations  of  food  and  drug 
contamination  on  the  part  of  a  few  of  the  more  enterprising  state 
and  municipal  health  ofhcers.  Very  recently  the  sanitary  study 
of  mineral  waters'  has  received  a  great  deal  of  attention  on  the 
part  of  individual  workers.  The  Committee  of  the  Laboratory 
Section  of  the  American  Public  Health  Association  has  prepared 
a  report  covering  the  general  conclusions  regarding  some  of  the 
methods  of  analysis. 

The  purely  microscopical  examination  of  food  substances  and 
of  drugs,  with  reference  to  contamination  by  mold,  yeast  and 
bacteria,  should  be  a  part  of  the  work  of  the  bacteriologist  rather 
than  that  of  the  chemist.  Therefore,  for  the  sake  of  completeness, 
this  phase  of  the  subject  is  included  in  the  present  report.  We 
shall  now  proceed  with  the  discussion  of  the  bacteriological  method 
applicable  in  food  and  drugs  laboratories,  giving  only  the  essential 
details,  however  adding  certain  suggestions  intended  as  a  guide 
for  further  investigation  with  a  view  to  the  improvement  of  the 
present  working  methods.  Detailed  description  of  apparatus  and 
of  technique  will  be  given  only  when  thought  necessary. 

For  all  practical  purposes,  the  examination  of  foods  and  drugs 
for  the  presence  of  biologic  contamination  (inclusive  of  bacteria, 
yeasts,  molds,  protozoa,  ova  and  larvae  of  higher  animal  parasites, 
etc.,  etc.)  is  either  made  directly  or  indirectly.  That  is,  the  sub- 
stance is  either  placed  on  a  slide  or  counting  apparatus  and  the 


lN'J\RODUCTION  35 

quantitative  or  qualitative  determinations  made  directly  under  the 
suitable  power  of  the  compound  microscope;  or,  certain  quantities 
of  the  substances  are  placed  in  or  upon  certain  culture  media 
(Petri  dish  cultures,  tube  cultures,  etc.)  in  order  to  bring  out  the 
biological  and  biochemical  characteristics  of  the  contaminating 
organisms,  whereupon  the  cultural  products  are  examined  micro- 
scopically. In  this  latter  instance  the  microscopical  examination 
may  even  be  entirely  omitted. 

The  direct  microscopical  method  has  some  very  marked  ad- 
vantages and  should  be  carried  out  whenever  feasible,  particularly 
when  purely  quantitative  results  or  estimates  is  the  main  object 
sought  after.  In  other  instances  the  direct  method  must  be 
combined  with  cultural  tests  and  the  two  are  often  checks  upon 
each  other. 

2.  Direct  Bacteriological  Examinations — Quantitative  Tests 

Substances  to  be  examined  include  waters  and  liquids  of  all 
kinds;  sewage;  milk^  and  cream;  ice  cream,  liquid  pharmaceuti- 
cals and  medicamenta,  oils,  catsups,  beverages  of  all  kinds,  all 
semisolids  such  as  pastes,  jams,  jelUes,  etc..  Other  semiHquids  and 
semisolids  which  may  be  readily  diluted  with  water  if  necessary; 
solids  as  powders,  pills,  tablets,  soils,  clays,  meats,  starches, 
dextrins,  flours,  meals,  dried  fruits,  dried  eggs,  dried  albumen, 
sugar,  etc.  In  fact  all  substances  which  are  in  any  way  liable  to 
contamination  by  micro-organisms. 

The  following  is  an  outline  of  the  methods  of  making  determina- 
tions of  the  number  of  organisms  in  food  and  drugs. 

a.  Substances  Requiring  Concentration. — Certain  substances 
which  contain  comparatively  few  micro-organisms,  as  drinking 
waters,  mineral  waters,  beverages  generally,  tinctures,  fluid  ex- 

^  In  the  case  of  milk,  the  centrifuge  is  first  used  to  separate  out  the  fat  as  much 
as  may  be  necessary  to  make  the  ready  counting  of  the  organisms  possible.  (See 
also  Chapter  on  Milk  Analysis.) 

4 


36  BACTERIOLOGICAL   METHODS 

tracts,  aquae,  etc.,  must  be  subjected  to  processes  which  will  con- 
centrate the  organisms,  as  by  passing  the  liquid  through  a  filter 
in  which  the  pores  are  sufficiently  small  to  leave  the  organisms 
behind,  as  for  example  a  Berkefeld  or  Chamberland  clay  tube. 
In  addition  to  the  filter,  the  centrifuge  will  be  found  useful  as  will 
be  explained  later. 

Any  liquid  containing  not  more  than  from  100  to  1,000,000 
organisms  per  cc.  does  not  lend  itself  to  direct  examination  quanti- 
tatively without  concentration.  The  amount  or  volume  of  sub- 
stance (liquid)  to  be  passed  through  the  filter  will  depend  upon  the 
degree  of  concentration  required.  Since  the  Thoma-Zeiss 
hemacytometer  (with  Turck  ruling)  is  to  be  used  in  making  the 
counts,  the  organisms  should  average  at  least  4  to  5  in  the  J-^so 
c.mm.  areas  of  the  counting  apparatus,  or  1,000,000  to  1,250,000 
organisms  per  cc.  Let  us  suppose  that  a  direct  count  is  to  be  made 
of  a  drinking  water  which  is  very  pure,  having  not  more  than  from 
50  to  500  bacteria  per  cc.  In  order  to  make  direct  counting  with 
the  hemacytometer  possible,  it  would  be  necessary  to  pass  from 
20  to  30  liters  of  the  water  through  the  clay  filter  and  thoroughly 
mix  the  organisms  left  in  the  tube  with  10  or  even  i  cc.  of 
filtered  sterile  water.  To  filter  that  amount  of  water  requires 
too  much  time  unless  a  large  specially  constructed  apparatus  is 
installed.  For  practical  purposes,  i  liter  is  the  largest  amount 
of  liquid  that  it  will  be  necessary  to  filter  and  reduce  to  i  cc, 
making  a  concentration  of  1000.  For  special  purposes  the  i  cc. 
may  be  further  concentrated  in  the  centrifugal  tube  described  in 
Fig.  3.  Weaker  concentrates  may  answer  the  purpose  in  some 
cases,  as  ten  or  one  hundred,  as  in  sewage,  badly  contaminated 
milk  and  in  other  liquids  in  which  the  number  of  organisms 
present  may  range  from  100,000  to  1,000,000  per  cc. 

The  special  centrifugal  tube  described  in  Fig.  3  is  used  as 
follows :  After  passing  a  liter  of  the  liquid  to  be  examined  through 
the  clay  filter  tube  and  thoroughly  washing  out  the  organisms  and 
other  particles  left  in  the  tube,  pour  the  contents  into  the  special 


DIRECT  EXAMINATION 


37 


---/Occ. 


Ice. 


B 


Fig.  2.  Fig   3. 

Fig.  2. — Kitasato  filtering  outfit  ready  to  be  attached  to  the  exhaust  pump. 
A  two-opening  flask  or  bottle  is  interpolated  to  receive  the  backflow  water,  should 
there  be  any.  Various  types  of  clay  bougies  may  be  used  with  this  filter.  The 
rubber  tubing  for  the  connection  must  be  heavy  so  as  to  prevent  collapse  by  the  ex- 
haust pressure. — {Pit field.) 

Fig.  3. — Special  centrifugal  tube  {A)  for  concentrating  bacteria  and  other  micro- 
organisms in  liquids  and  also  used  in  isolating  or  separating  motile  bacteria  from 
those  which  are  not  motile,  as  is  explained  under  water  analysis.  The  tube  has  a 
capacity  of  15  cc.  with  i  cc.  and  10  cc.  marks.  The  tube  is  in  two  parts.  The  lower 
narrowed  end,  having  a  capacity  of  i  cc,  is  attached  to  the  larger  part  by  means  of 
a  rubber-band  ring.  CentrifugaHzation  is  done  at  high  speed.  After  centrifugali- 
zation,  the  i  cc.  tube  is  removed  and  the  contents  thoroughly  mixed  by  means  of  a 
platinum  wire  loop.  To  avoid  loss  of  the  contents  of  the  tube  during  the  mixing, 
attach  the  rubber  ring.  After  the  mixing  the  material  is  ready  for  the  microscopical 
counting  and  other  examination. 

A  suitable  stopper  attached  to  a  brass  or  other  metal  rod  {B)  may  be  inserted 
into  the  narrowed  portion  of  the  upper  part  of  the  tube  in  order  to  prevent  mixing 
of  contents  when  removing  the  i  cc.  tube.  These  tubes  will  also  be  found  useful 
in  measuring  the  amount  of  sediment  in  milk,  water  and  other  liquids.  For  this 
purpose  the  i  cc.  portion  should  be  graduated  into  tenths  and  hundredths. 


38  BACTERIOLOGICAL  METHODS 

centrifugal  tube.  For  washing  use  about  lo  cc.  of  filtered  sterile 
water,  adding  up  to  the  lo  cc.  mark  if  necessary.  Centrifugalize 
at  high  speed  for  30  min.,  which  will  throw  the  bacteria  and  other 
solids  down  into  the  narrow  i  cc.  end  of  the  tube. 

The  following  is  a  brief  outline  of  the  method  of  procedure: 
Use  a  Kitasato  filter  with  the  usual  hydrant  suction  pump  attach- 
ment. Pass  a  liter  of  the  liquid  to  be  examined  through  the 
filter,  continuing  suction  until. nearly  all  of  the  liquid  has  passed 
through.  Remove  the  clay  bougie  and  wash  down  the  organ- 
isms clinging  to  the  sides  of  the  tube  with  not  more  than  10  cc. 
of  distilled  water  which  has  been  filtered  and  boiled.  Place  thumb 
over  the  opening  of  the  tube  and  mix  the  contents  thoroughly  by 
shaking  for  20  sec,  then  pour  the  thoroughly  mixed  contents 
into  a  sterile  cylindrical  graduate  and  add  sterile  distilled  and 
filtered  water  up  to  the  10  cc.  mark,  shake  thoroughly  and  make 
the  counts  at  once  by  means  of  the  hemacytometer.  This  pro- 
cedure gives  a  concentration  of  100.  By  means  of  the  special 
centrifugal  tube  the  concentration  may  be  increased  to  1000, 
as  already  explained.  The  method  gives  approximate  results 
only,  the  counts  as  a  rule  being  less  than  the  actual  number  of 
organisms  present  in  the  liquid,  a  difference  due  to  three  chief 
sources  of  error:  First,  a  small  number  of  bacteria  (especially 
the  smaller  motile  forms)  will  pass  through  the  clay  filtering  tube; 
second,  some  of  the  smaller  bacteria  are  caught  and  held  in  the 
pores  of  the  clay  tube;  and  third,  some  organisms  will  remain 
clinging  to  the  inner  surface  of  the  tube  after  the  mixed  contents 
are  poured  out  for  counting  purposes.  These  sources  of  error 
are,  however,  not  great,  perhaps  not  exceeding  8  to  10  per  cent., 
and  are  on  the  side  of  conservative  estimates.  The  clay  bougies 
used  should  be  of  the  finest  quality  and  should  be  of  uniform  and 
standard  thickness.  The  sources  of  error  by  the  direct  method 
are  perhaps  not  as  great,  certainly  not  greater,  than  by  the  usual 
plating  methods  and  offer  sorne  very  decided  advantages.  The 
concentrates  show,  in  addition  to  the  bacteria,  other  organisms 


DIRECT  EXAMINATION 


39 


Fig.  4. — Apparatus  for  fractional  filtration,  designed  for  use  with  Pasteur- 
Chamberland  or  Berkefeld  filters,  a,  Glass  mantle  surrounding  filter;  b,  Chamber- 
land  filter;  c,  paraffin  joint;  d  and  e,  rubber  stoppers;  /,  double  side-arm  suction 
flask;  g,  pinchcock  controlling  outlet  from  suction  flask;  A,  outlet  tube  surrounded 
by  glass  shield  and  attached  to  lower  end  of  suction  flask  by  means  of  short  rubber 
tubing;  i,  glass  shield  fused  to  and  surrounding  outlet  tube  as  ^  protection  against 
contamination  when  the  filtrates  are  drawn  off;  j,  glass  inlet  tube  plugged  with  cotton, 
for  admitting  air  into  suction  flask;  k,  pinchcock  governing  the  admission  of  air 
into  flask;  I,  vacuum  gauge;  m,  stopcock  connected  with  vacuum  pump. — (U.  S 
Dept.  of  Agriculture,  Bureau  of  Animal  Industry,  Bull.  113.) 


40  BACTERIOLOGICAL   METHODS 

as  mold  h3^hae,  mold  spores,  protozoa,  diatoms,  etc.,  besides 
dirt  particles,  sand  particles,  starch  granules,  body  cells,  pus 
cells,  etc.,  etc.,  which  would  be  lost  or  rather  which  would  not 
appear  in  the  plating  method.  Furthermore,  the  counts  can  be 
made  with  a  great  saving  of  time — in  a  few  hours  as  against  24 
to  48  hr.,  and  longer,  by  the  plate  cultural  method.  It  is  true 
that  in  many  instances  the  direct  method  must  be  supplemented 
by  the  cultural  methods  when,  in  the  judgment  of  the  analyst, 
this  becomes  desirable  or  necessary. 

Concentrates  may  also  be  made  by  evaporation  under  reduced 
pressure.  With  a  little  ingenuity  a  suitable  equipment  may  be 
constructed  in  the  laboratory.  The  container  of  suitable  ca- 
pacity (i  liter  and  more)  is  connected  with  an  exhaust  pump 
which  lowers  the  pressure  sufficiently  to  cause  boiling  at  a  tem- 
perature not  to  exceed  37°  C;  24  hr.  is  usually  sufficient  time 
to  evaporate  the  liquid  to  nearly  dryness.  After  the  evaporat- 
ing process  has  continued  for  several  hours,  various  enrichment 
media  may  be  added  to  the  liquid  to  be  evaporated,  which  will  of 
course  aid  the  intended  isolation  and  development  of  the  de- 
sired bacteria.  If  the  enrichment  medium  is  added  from  the 
first,  annoying  bubbling  and  frothing  may  take  place.  This 
method  is  especially  useful  in  isolating  the  typhoid  bacillus,  the 
paratyphoid  group  and  the  intestinal  bacteria  in  general. 

b.  Substances  Which  do  not  Require  Concentration. — Badly 
contaminated  substances  as  sewage,  milk  from  badly  managed 
dairying  establishments,  badly  contaminated  liquids  of  all  kinds, 
soups,  broths,  beer,  wines  and  such  products  as  tomato  catsups, 
jams,  jellies,  canned  oysters,  etc.,  may  be  examined  directly 
without  the  necessity  of  making  concentrations,  or  of  centrifugali- 
zation,  in  order  to  make  quantitative  and  certain  qualitative 
estimates.  The  substances  of  this  class  may  be  divided  as  follows, 
based  upon  the  approximate  number  of  organisms  per  cc.  as 
determined  by  means  of  the  spore  and  yeast  counter  described 
under  Fig.  5,  and  the  Thoma-Zeiss  hemacytometer. 


DIRECT  EXAMINATION 


41 


COUNTING 

APPARATUS 

for 

MOLDS  aWSPORES 


1                     1 

?                 ZS                so                75 

1 

■ 

1 

1 

■ 

1 

1 

1 

1 

1 

1 

1 

1 

I 

1 

05                   10                IS 

1                                             1 

AREAS 

'/zssq.m.m. 

tsqmm. 
25  5<7  mm. 

15s<{.mm. 

CUBIC  CONTENTS 
'/us  cmm. 
'/sc.mm. 
sc.mm. 
15  cmm. 


A 

/    2   3   4-56 

B 

12    3    4-56 

a 
b 

a 
b 

c 

D 

Fig.  5. — A,  Counting  apparatus  for  molds  (hyphae  and  spores)  and  yeasts.  The 
rulings  are  75  sq.  mm.,  25  sq.  mm.,  i  sq.  mm.  and  3^5  sq.  mm.  On  either  side  of  the 
ruled  area  are  glass  slips  0.2  mm.  thick,  so  that  the  entire  capacity  of  the  space 
within  the  ruled  area  is  15  cmm.,  subdivided  into  s  cmm.,  }^  cmm.  and  ^25 
cmm.  areas. 


42  BACTERIOLOGICAL   METHODS 

1.  Substances  in  which  the  organisms  are  not  too  numerous  to 
permit  the  use  of  the  3^5  sq.  mm.  areas  without  making  dilutions. 
That  is,  substances  in  which  the  number  of  organisms  does  not 
exceed  10,000,000  per  cc,  hence  the  number  of  yeast  cells,  spores, 
bacteria,  etc.,  may  not  exceed  forty  in  one  of  the  3^50  c.mm.  areas 
of  the  hemacytometer.  The  limit  for  the  spore  and  yeast  counter 
would  be  5,000,000,  before  making  the  dilution  is  necessary. 

2.  Substances  in  which  the  number  of  organisms  and  spores 
are  too  numerous  to  permit  the  use  of  the  J-^s  sq.  mm.  areas  of  the 
hemacytometer,  but  permitting  the  use  of  the  }ioo  sq.  mm.  areas 
without  making  dilutions.  The  total  number  of  spores,  bacteria 
and  other  organisms  may  range  from  10,000,000  to  100,000,000 
per  cc,  numbers  derived  from  finding  on  an  average  from  2.5  to 
25  organisms  in  one  of  the  34ooo  c.mm.  areas  of  the  hemacytometer. 

The  counter  is  used  as  follows:  A  bit  of  the  thoroughly  mixed  substance,  as 
jam,  jelly,  tomato  paste,  catsup,  etc.,  is  placed  on  the  slide  in  the  ruled  areas  and 
covered  with  a  rectangular  cover  glass  (No.  2).  Slight  pressure  may  be  necessary 
to  make  the  cover  glass  rest  evenly  on  the  two  slips.  The  counting  is  done  in  areas 
entirely  filled  (from  slide  to  cover  glass)  by  the  substance  mounted.  The  larger 
areas  may  prove  useful  in  estimating  the  amount  of  sand  particles,  dirt,  etc.,  present. 
The  K25  c.mm.  areas  will  be  used  in  counting  spores,  yeast  cells  and  mold  hyphae 
and  similar  contaminations.  It  is  possible  to  make  counts  without  dilutions  as 
long  as  the  number  of  organisms  in  the  areas  does  not  exceed  forty.  If  more 
organisms  are  present  in  one  area  dilution  becomes  necessary,  as  already  ex- 
plained. Making  dilutions  of  1-10,1-100  and  i-iooo  makes  the  counting  limits 
50,000,000,  500,000,000  and  5,000,000,000  per  cc.  The  3^25  c  mm.  areas  are  also 
used  in  estimating  the  quantity  of  mold  hyphae  present.  Finding  clusters  of  mold 
hyphae  in  25  per  cent,  of  these  smallest  areas  is  presumptive  proof  that  the  substance 
is  unfit  for  human  consumption.  Naturally  the  more  finely  divided  the  substance 
is  the  more  numerous  are  the  mold  fragments.  For  making  mold  counts  the  material 
to  be  examined  should  be  reduced  to  uniform  fineness.  This  could  be  accomplished 
by  rubbing  a  thoroughly  mixed  sample  through  a  sieve  of  standard  mesh,  say  3<4  mm. 

B,  a  simplified  modification  of  the  counting  apparatus  just  described,  is  made  as 
follows:  The  two  slips  0.2  mm.  thick  are  placed  in  position,  but  the  ruling  is  omitted 
and  in  place  thereof  an  eye-piece  scale  C  is  used,  the  measuring  value  of  which  has 
been  carefully  determined  by  means  of  the  stage  micrometer.  The  rulings  on  the 
eye-piece  must  be  delicate  and  the  analyst  must  be  careful  not  to  move  the  eye  or 
change  the  direction  of  his  vision  while  making  counts. 

The  ruled  slide  (Z>)  will  be  found  useful  for  making  quantitative  estimates  of 
seeds,  sand  particles,  dirt,  larger  parasites  such  as  vinegar  eels,  ova  of  intestinal 
parasites,  etc.,  in  catsups,  crushed  berries  (strawberries,  raspberries,  loganberries, 
etc.),  jams  and  in  other  vegetable  substances.  Definite  quantities  of  the  substance 
to  be  examined  are  placed  upon  the  ruled  area  of  the  slide  by  means  of  a  small 
measuring  spoon  (0.25  gram,  0.5  gram,  i  gram),  spread  and  covered  with  a  suitable 
cover  glass  and  the  counts  made  in  the  entire  amount  placed  on  the  slide,  using  the 
low  power  (80  diam.)  of  the  compound  microscope. 


DIRECT   EXAMINATION  43 

3.  Substances  in  which  the  organisms  are  too  numerous  to 
permit  ready  counting  by  means  of  the  0.004  c.mm.  areas  of  the 
Thoma-Zeiss  hemacytometer.  It  now  becomes  necessary  to  use 
dilutions,  which  are  made  as  follows. 

Making  the  Dilutions. — The  dilutions  generall)^  used  are  i-io. 
Rarely  will  it  be  found  necessary  to  use  higher  dilutions.  Should 
this,  however,  become  desirable,  a  dilution  of  i-ioo  is  to  be  made. 
The  highest  counts  so  far  recorded  were  in  the  case  of  two  tomato 
pastes  which  showed  2,400,000,000  and  4,000,000,000  bacilli  per 
cc.  In  these  instances  dilutions  of  i-io  were  used  and  proved 
quite  satisfactory,  though  it  was  evident  that  a  greater  number  of 


Fig.  6. — Thoma-Zeiss  hemacytometer.  Complete  equipment  for  blood  count- 
ing. This  is  very  convenient  for  making  bacterial  counts  in  catsups,  jams,  jellies 
and  other  vegetable  foods  and  also  in  animal  food  substances. 

bacilli  per  cc.  would  have  necessitated  the  use  of  a  dilution  of 
i-ioo.  However,  a  dilution  of  i-io  is  all  that  is  required  for 
practical  purposes,  as  a  bacterial  count  of  4,000,000,000  and  more 
per  cc.  would  indicate  the  decomposed  condition  of  the  food 
substance  and  its  unfitness  for  human  consumption. 

In  case  of  liquids  and  near  liquids,  9  cc.  of  distilled  water  is 
added  to  i  cc.  of  the  substance,  and  in  the  case  of  pastes  and 
similar  products,  9  cc.  of  distilled  water  is  added  to  i  gram  (or 
I  cc.  semiliquid)  of  the  substance.  The  dilutions  are  made  in 
25  cc.  graduated  cylinders,  which  answer  the  purpose  very  well. 
Or  100  cc.  graduates  may  be  used  for  making  the  dilutions,  adding 


44 


BACTERIOLOGICAL  METHODS 


90  cc.  of  distilled  water  to  10  grams  (or  10  cc.)  of  the  substance. 
Place  the  thumb  firmly  over  the  opening  of  the  graduate;  the  con- 
tents  are  thoroughly  mixed  by  shaking  vigorously  for  about  20  sec. 
By  means  of  a  slender  glass  rod  slipped  well  into  the  mixture,  take 
up  a  droplet  of  the  mixed  material  and  touch  the  end  of  the  rod 
lightly  and  quickly  upon  the  middle  of  the  ruled  area  of  the  hem- 
acytometer.    All  this  must  be  done  rapidly,  before  the  organisms 


Fig.  7.  Fig.  8. 

Fig.  7. — Zappert  ruling  of  the  Thoma-Zeiss  hemacytometer.  This  form  of 
ruling  is  especially  convenient  for  making  bacterial  counts  and  counts  of  fat  globules 
in  milk. — {Carl  Zeiss.) 

Fig.  8. — Turck  ruling  of  the  Thoma-Zeiss  hemacytometer.  This  is  especially 
useful  if  it  is  desired  to  combine  the  bacterial  count  with  the  spore  and  yeast  count. 
The  smaller  areas  (1-400  sq.  mm.)  may  be  used  for  making  the  bacterial  counts,  while 
the  larger  areas  (1-25  sq.  mm.)  may  be  used  for  making  the  spore  and  yeast  counts. — 
{Carl  Zeiss.) 

have  had  time  to  settle  to  the  bottom  of  the  graduate,  and  before 
they  have  had  time  to  accumulate  at  the  end  of  the  glass  rod. 

Making  the  Cotint.^ — ^After  having  cleaned  the  hemacytometer 
(do  not  use  alcohol),  it  is  sometimes  desirable  to  rub  a  very  soft, 
grit-free  graphite  pencil  over  the  ruled  area  so  as  to  render  the 
lines  more  readily  visible.  Usually,  however,  this  is  not  necessary. 
After  placing  the  droplet  of  material  as  above  described,  cover 
with   a   No.    2    cover  glass   and   orientate   the   ruled    area   by 


DIRECT   EXAMINATION 


45 


means  of  the  low  power  and  make  counts  under  the  suitable  high 
DOwers.  From  ten  to  twenty  of  the  ruled  areas  should  be  counted 
md  from  these  countings  figure  the  average.  It  is  desirable  to 
make  two  or  three  mounts  of  each  sample,  thus  giving  the  average 
3f  from  twenty  to  thirty  areas  counted.  The  countings  are  to 
De  made  in  areas  free  from  pulp 
Fragments  and  including  all  organ- 
sms  lying  within  the  ruled  bound- 
ng  lines  and  inclusive  of  half 
averages  of  those  organisms  which 
ie  across  the  rulings.  All  count- 
ings which  present  characters  of 
doubt  are  omitted  from  the  final 
estimates. 

Those  organisms  which  occur 
within  the  cell-lumen  of  the  vege- 
table tissues  are  not  to  be  counted. 
To  do  so  is  practicably  impossible 
and  such  countings,  even  if  pos- 
sible, would  add  nothing  to  the 
value  of  the  findings.     In  case  the 

cells  contain  numerous  bacteria  this  should  be  noted  in  the  report, 
as  it  certainly  indicates  decomposition  of  the  material.  The  prin- 
cipal decomposition  changes  due  to  the  invasion  of  bacteria  and 
other  organisms  are,  however,  largely  limited  to  the  exterior  of 
cells,  especially  by  those  organisms  which  develop  during  or  after 
the  factory  processing.  The  numerical  determinations  are  there- 
fore limited  to  organisms  which  occur  in  the  matrix  and  those  which 
have  been  washed  from  the  exterior  of  cells  by  the  thorough  mixing. 
The  thorough  mixing  of  the  samples  is  a  very  important  part  of 
the  procedure.  In  the  case  of  liquids  and  semiliquids,  mixing  is 
done  by  thorough  shaking,  aftd  in  the  case  of  pastes  and  similar 
materials,  by  means  of  a  spatula  or  a  small  spoon. 

In  making  counts  of  very  small  or  comparatively  short  bacilli, 


Fig.  9. — Biirker  ruling,  useful  in 
making  counts  of  milk  fat  globules, 
spores,  and  yeast  cells.  The  average 
of  many  counts  is  taken. — {Carl  Zeiss.) 


46 


BACTERIOLOGICAL   METHODS 


some  difficulty  is  caused  by  those  organisms  which  happen  to  be 
vertically  suspended  in  the  counting  chamber,  thus  presenting  an 
end  view  which  gives  the  appearance  of  small  granules  or  spherical 
particles  which  the  comparatively  inexperienced  observer  may  not 
recognize,  or  which  may  be  mistaken  for  inorganic  particles  or 
organic  particles  other  than  microbic.     In  case  of  doubt,  allow  the 


■ 

^^— ^ 

^^^^^Kf^^" 

■p-^ 

i 

r^ 

1 

^^^^^^^P^/ 

V" 

■■>,  %■ 

\( 

'        / 

Fig.  io. — Tomato  pulp  cells  in  normal  catsup.  The  cells  are  large,  thin-walled, 
containing  granular  particles.  The  coloring  matter  of  the  tomato  frequently  ap- 
pears as  deep  scarlet-red  crystalline  particles  usually  arranged  in  groups  within  the 
cell. — {Howard,  Yearbook  U.  S.  Dept.  oj  Agriculture,  191 1.) 

mount  to  remain  at  rest  for  10  or  15  min.,  thus  allowing  the 
bacilli  to  settle  to  the  bottom  of  the  cell  where  they  will  assume 
the  horizontal  position,  thus  presenting  the  long  axis  to  view 
and  making  counting  easier. 

In  order  that  all  of  the  cells  (individuals)  of  the  bacilli  may  be 
counted,  it  is  necessary  to  use  a  high  power  (480  to  500  diam.). 
Lower  powers  are  not  satisfactory  for  counting  bacteria.     Foi 


DIRECT   EXAMINATION 


47 


:ounting  spores  and  yeast  cells  a  magnification  of  i8o  diam. 
Yould  prove  very  satisfactory,  especially  with  a  well -corrected 
vide  aperture  objective.  The  counting  of  cocci  is  more  confusing 
:han  the  counting  of  bacilli,  but  fortunately  the  microbic  contami- 


FiG.  II. — Cluster  of  mold  hypba;  in  granular  (decomposed)  tomato  pulp.  This 
type  of  mold  is  traceable  to  field-rotted  tomatoes.  The  finding  of  hyph^  of  this 
type  in  tomato  catsup  indicates  the  use  of  rotted  tomatoes,  therefore,  indicates 
inadequate  culling  at  the  isictory.— {Howard,  Yearbook  U.  S.  Dept.  of  Agriculture, 
1911.) 


nations  of  most  vegetable  substances  are  bacillar,  though  there  are 
some  notable  exceptions. 

Mold  Counting.— Thus  far  no  satisfactory  method  for  making 
estimates  of  the  amount  of  mold  hyphae  present  in  fruit  and  in 
animal  products  has  come  into  use.  The  method  recommended 
by  B.  J.  Howard,  Chief  of  the  Micro-chemical  Laboratory  of  the 
U.  S.  Bureau  of  Chemistry,  namely,  determining  the  degree  of 


48  BACTERIOLOGICAL   METHODS 

mold  contamination  from  the  number  of  microscopic  fields  of  the 
compound  microscope  which  show  the  presence  of  hyphal  clusters, 
is  far  from  satisfactory.  It  indicates  the  amount  of  contamination 
in  a  general  way  only.  More  rehable  and  more  accurate  estimates 
could  be  obtained  through  the  use  of  a  counting  apparatus  in  which 
the  number  of  hyphal  clusters  could  be  ascertained  in  definite 
quantities  of  the  material  under  examination.  The  hemacy- 
tometer already  mentioned,  does  not  serve  the  purpose  because  of 
the  smallness  of  the  counting  areas.  The  special  counter  described 
in  Fig.  5  would  serve  the  purpose  very  well.  It  is  furthermore 
necessary  to  reduce  the  material  to  a  uniform  and  standard  fineness 
by  rubbing  it  through  a  sieve.  A  very  small  standard  mesh  sieve 
would  answer  the  purpose.  Take  i  gram  of  the  thoroughly  mixed 
material  and  by  means  of  a  small  spatula  rub  all  of  it  through  the 
sieve  and  make  the  estimations  from  the  pulp  which  has  been 
passed  through  the  meshes  of  the  sieve. 

Precautions. — The  following  are  some  of  the  factors  which 
necessitate  caution  in  making  counts  of  microbes,  yeast  cells, 
spores  and  mold  fragments. 

a.  Badly  decomposed  factory  pulp  which  compels  prolonged 
heating  in  order  to  render  it  suitable  for  canning,  often  presents 
such  a  granular  appearance  as  to  make  accurate  counting  of  the 
microbes  rather  difficult.  In  such  materials  many  of  the  more 
or  less  disintegrated  pulp  cells  are  filled  with  bacteria  and  these 
cannot  be  included  in  the  count.  Commonly  in  such  substances 
many  of  the  mold  fragments  are  also  very  much  disintegrated 
through  decomposition  changes,  probably  initiated  by  enzymes 
formed  by  the  bacteria  and  other  organisms. 

h.  While  it  is  quite  easy  to  distinguish  between  living  yeast 
cells,  dead  yeast  cells  and  spores,  it  is  not  thought  advisable  to 
attempt  such  differentiation  in  routine  laboratory  practice,  ex- 
cepting in  cases  where  identification  is  simple  and  where  there  is 
very  little  room  for  doubt.  One  of  the  first  important  problems 
for  the  food  and  drugs  bacteriologist  to  solve  is  the  identification 


DIRECT  EXAMINATION  49 

of   those   micro-organisms    which    commonly  attack   foods  and 
drugs,  more  especially  the  molds  and  yeasts. 

c.  It  is  neither  practicable  nor  necessary  to  differentiate 
between  the  different  kinds  of  spores  which  may  be  present  in  a 
product,  excepting  as  suggested  under  (6). 

d.  In  many  instances  it  would  be  desirable  to  resort  to  plat- 
ing methods  in  order  to  determine  the  number  of  viable  organ- 
isms present.  This  would  be  simple  for  bacteria  and  mold  spores, 
but  more  difficult  for  yeasts. 

Differentiating  between  Living  and  Dead  Bacteria  and  other 
Low  Forms  of  Organisms. — It  would  be  most  desirable  to  deter- 
mine some  practical  working  method  for  distinguishing  between 
living  and  dead  bacteria  in  foods  and  drugs.  Often  the  question 
arises  as  to  the  time  and  place  source  of  the  contamination.  Did 
the  organisms  present  develop  in  the  fruit,  in  the  pulped  material 
during  the  processing  or  in  the  containers  after  manufacture? 
Again,  are  the  organisms  estimated  by  the  direct  count  dead  or 
alive? 

Several  investigators  have  stated  that  dead  and  living  bacteria  ^ 
react  differently  with  certain  stains.     For  example  G^Brqca,  an  I  ^^ 
Italian  bacteriologist,  declares  that  the  use  of  the  following  mixed 
stain  will  serve  this  purpose.     To  8  cc.  of  concentigte^  jrarb^ol^  ^  ^ 
fuchsin  add  loo  cc.  of  Loeffier's  methylene  blue-*^"Let  the  mixture       ' 
stand  for  24  hr.  before  using.     Exposed  to  this  stain,  dead  bacteria 
(killed  by  heat  or  by  disinfectants)  are  colored  red  while^  living 
bacteria  are  colored  blue.     It  is  declared  that  other  stains,  as  ^ 
Giemsa's,  will  react  in  a  similar  manner. 

More  recent  experiments  would  indicate  that  selenium  and 
tellurium  compounds  will  serve  to  differentiate  Uving  bacterial 
contaminations.  It  would  appear  that  these  substances  are 
decomposed  into  metallic  tellurium  and  selenium  when  brought 
in  contact  with  living  organisms.  Much  experimental  work 
along  this  line  has  been  done  by  Hansen,  Gmelin,  Gosio  and 
others,  and  still  more  recently  (1913)  by  King  and  Davis  of  the 


50  BACTERIOLOGICAL   METHODS 

ResearchLaboratory  of  Parke,  Davis  and  Company.  Potassium 
tellurite  is  said  to  be  the  most  satisfactory  reagent.  In  dilutions 
of  I  :  50,000  this  substance  forms  characteristic  black  compounds 
with  all  of  the  more  common  micro-organisms  when  in  the  living 
state.  The  reaction  does  not  take  place  in  the  presence  of  dead 
micro-organisms  and  the  different  organisms  do  not  all  react  in 
the  same  degree  or  manner.  Some  are  much  more  susceptible 
than  others.  The  Bacillus  coli  appears  to  be  the  most  sensitive 
to  the  reagent.  With  most  species  of  bacteria  the  time  re- 
quired to  produce  the  characteristic  color  and  precipitation  reac- 
tion ranges  from  12  to  96  hr.  at  a  temperature  of  37°  C,  but  with 
the  colon  bacillus  a  distinct  coloration  or  color  ring  becomes  visible 
several  minutes  after  the  reagent  is  added.  King  and  Davis 
summarize  the  experimental  results  as  follows: 

1.  Nearly  all  of  the  more  common  micro-organisms  (bacteria  and  yeasts)  react 
with  potassium  tellurite,  forming  characteristic,  black  compounds. 

2.  This  capacity  depends  on  an  active  stage  of  metabolism  of  the  reacting 
organism,  and  the  action  is,  in  all  probability,  a  reduction  of  the  tellurite, 

3.  The  "tellurite  reaction"  can  be  used  as  an  indicator  of  microbial  life,  and  is 
especially  suitable  for  revealing  microbic  contamination. 

4.  A  dilution  of  i  :  50,000  of  the  salt  seems  to  be  most  suitable  for  its  action  as  a 
general  microbic  indicator.  In  this  concentration,  it  produces  no  irritative  action 
when  introduced  into  test  animals. 

5.  The  bacteria  of  the  " colon- typhoid  group"  show  dififerences  in  resistance 
to  the  antiseptic  action  of  potassium  tellurite  and  in  the  appearance  of  their  reaction 
with  this  salt.  These  variations  are  sufficient  to  suggest  the  experimental  use  of 
potassium  tellurite  for  differential  diagnosis  in  the  group. 

6.  The  intensity  of  bacterial  action  on  potassium  tellurite  depends  upon  the 
individual  resistance  of  the  bacterium  and  the  concentration  of  the  salt  present. 
The  velocity  of  reduction  of  the  tellurite  is  apparently  a  specific  function  of  an  organ- 
ism, apart  from  its  resistance  to  antiseptic  action.  With  the  colon  bacillus,  the 
"tellurite  reaction"  is  almost  instantaneous. 

7.  Treatment  with  potassium  tellurite  has  practically  no  influence  on  the  bio- 
logical characteristics  of  an  organism. 

3.  Numerical  Limits  of  Micro-organisms  in  Foods  and  Drugs 

What  should  be  the  maximum  limit  of  the  number  of  bacteria 
and  other  micro-organisms  in  food  and  drugs  within  the  intent 


I 


DIRECT  EXAMINATION 


51 


3f  the  U.  S.  Pure  Food  and  Drugs  Act?  This  is  as  yet  an  un- 
settled question  and  one  that  requires  further  careful  considera- 
tion, even  calling  for  some  extensive  investigation  in  order  that 
:ertain  disputed  points  may  be  finally  settled.  There  are, 
lowever,  certain  results  based  upon  extensive  observation  which 


Fig.  12. — Type  of  mold  develcrpment  in  the  tomato  pulp  during  and  after  the 
processing.  According  to  tests  made  by  B.  J.  Howard  of  the  Bureau  of  Chemistry, 
mold  will  develop  in  tomato  catsup  containing  o.i  per  cent,  sodium  benzoate.  Com- 
pare the  hyphse  with  those  shown  in  Fig.  11.  They  are  much  larger  m  transverse 
diameter  and  the  walls  of  the  cells  are  much  thinner.— {Bitting,  Bull.  119,  Bureau 
of  Chemistry,   U.  S.  DepL  of  Agriculture.) 

may  be  set  down  as  conclusive.  The  organisms  of  all  kinds  which 
may  occur  in  and  upon  clean  and  uncontaminated  ripe  fruit, 
for  example,  are  negligible  quantitatively  as  well  as  quaUtatively. 
Such  organisms  as  do  occur  are  limited  to  the  exterior.  Only 
under  abnormal   conditions  do  micro-organisms  find  their  way 


52  BACTERIOLOGICAL  METHODS 

into  the  tissues  beneath  the  epidermis  and  into  the  parenchyma- 
tous cells  of  whole  fruits.  It  would  be  interesting  to  determine 
the  average  number  of  bacteria  on  the  exterior  of  such  fruits  as 
the  apple,  the  peach,  the  pear,  the  apricot,  the  tomato,  the 
cucumber,  etc.,  and  from  these  figures  to  estimate  the  number  of 
organisms  per  cc.  of  the  fruit  substance.  The  practical  value  of 
such  information  would,  however,  not  be  great,  as  may  be  understood 
from  the  statements  already  made.  It  must  be  admitted  without 
question  or  doubt  that  fruit  products  of  any  kind,  which  contain 
only  such  organisms  as  normally  occur  on  clean  uncontaminated 
ripe  fruit,  will  never  come  under  the  ban  of  the  pure  food  and 
drugs  act.  This  also  applies  to  foods  and  drugs  in  general.  The 
organisms  which  concern  the  analyst  are  those  which  occur  in  and 
upon  contaminated  and  diseased  fruits  and  those  which  are  in- 
troduced or  added  or  allowed  to  develop  and  multiply  during 
the  processing,  and  afterward.  We  may  therefore  make  the 
following  postulate:  All  fruit  products  from  clean  uncontami- 
nated fruit  (ripe  or  green),  prepared  under  modern  sanitary  con- 
ditions, contain  micro-organisms  in  negligible  quantities  only. 
It  is  true  that  the  ideal  conditions  implied  in  this  postulate  may 
not  always  be  attained  in  practice,  yet  we  are  warranted  in 
making  a  second  postulate,  namely:  that  the  number  of  organ- 
isms present  in  fruit  products,  over  and  above  the  negligible 
quantities  mentioned,  are  in  direct  proportion  to  the  careless- 
ness in  the  various  steps  of  the  processing.  Stating  it  conversely, 
as  the  manufacturers  of  food  products  attain  the  practically  ideal 
conditions,  the  number  of  organisms  in  their  products  will  become 
gradually  negligible.  That  such  conditions  are  attainable  is 
clearly  shown  by  the  canned  products  of  the  careful  housewife  and 
of  the  careful  manufacturer.  What  may  be  done  by  the  careful 
housewife  may  be  done  even  better  by  the  careful  manufacturer, 
because  the  latter  can  employ  the  most  approved  modern  methods, 
aided  by  special  machinery,  which  are  not  at  the  disposal  of  tl 
housewife  or  even  of  the  small  manufacturer. 


of  th^ 

J 


DIRECT  EXAMINATION 


53 


In  a  general  way,  the  number  of  micro-organisms  in  food  prod- 
ucts and  in  liquids  intended  for  internal  use,  not  including  the  fer- 
mented products,  is  negligible  when  they  do  not  exceed  250,000 
per  cc.  (ranging  from  5000  per  cc.  to   the  maximum).     In  fer- 


FiG.  13. — Various  stages  in  the  germination  of  spores  in  catsups.  Note  trans- 
verse septation  and  branching  of  the  hyphae.  Germinating  spores  may  be  traceable 
to  the  tomato  from  the  field  or  they  may  be  from  spoiling  factory  pulp. — (Biliing, 
Bull.  119,  Bureau  of  Chemistry,  U.  S.  Dept.  oi  Agriculture.) 

mented  products,  as  cider,  vinegar,  wines,  beer,  etc.,  the  number 
of  organisms  present  may  be  much  greater,  but  even  here  the 
quantitative  estimates  generally  become  negligible  if  the  modern 
methods  of  purifying  or  clarifying  (through  sedimentation,  the 
use  of  albumen,  gelatin,  casein,  etc.),  filtration,  centrifugalization, 
and  sterilization  are  carried  out.     Of  course,  in  such  products  as 


54 


BACTERIOLOGICAL  METHODS 


sour  milk,  ripened  cream,  ripened  cheese,  sauerkraut,  pickles, 
etc.,  the  processes  of  clarification  are  not  applicable,  and  hence  we 
always  find  a  large  number  of  certain  predominating  types  or 
species  of  organisms  present. 

The  microscopical  examination  of  products  which  have  under- 
gone normal  fermentation  shows  that  the  number  of  organisms 
present  is  quite  variable,  depending  upon  a  variety  of  causes  and 
conditions.     This  can  readily  be  ascertained  from  the  examination 


^^^%.o 


Fig.  14. — Substances  frequently  found  in  tomato  catsup,  a,  Heat  dextrinized 
corn  starch.  Starch  is  frequently  used  as  a  filler  or  stiffening  agent,  b,  bacteria 
which  frequently  appear  in  great  numbers,  c,  Vinegar  eels  derived  from  cider  or 
wine  vinegar.  Soil  nematodes  may  also  be  found,  indicating  gross  soil  contamina- 
tion and  inadequate  washing  at  the  cannery,  d,  Nematode  iarvse  derived  from  the 
soil,     e,  f,  g,  h,  Spore  types  frequently  met  with  in  catsups,     i,  Yeast  cells. 

of  such  common  household  products  as  vinegar,  sour  cream,  cideiP 
apple  butter,  sour  milk,  etc.     It  would  be  most  desirable  to  de- 
termine the  exact  identity  of  the  organisms  which  produce  tfafl 
most  favorable  fermentation  changes  in  fermented  food  product^ 
This  has  been  done  in  some  cases  and  pure  cultures  of  the  specific 
organisms  are  used  for  manufacturing  purposes,  resulting  in  i. 
production   of  superior  food   articles.     When   the   fermentatii 


1 


DIRECT   EXAMINATION 


55 


processes  are  left  to  nature  the  result  is  not  by  any  means  uniform 
and  we  have  products  which  are  often  so  vitiated  by  the  develop- 
ment of  undesirable  associated  organisms  as  to  make  the  food  unfit 
for  use.  There  is  a  definite  biological  relationship  between  those 
organisms  which  initiate  desirable  fermentations  and  those  which 
are  objectionable;  both  kinds  are  generally  present,  but  fortunately 


Fig  15.— Vinegar  eels  from  decomposed  blackberry  pulp.  The  smaU  particles 
scattered  through  the  field  are  yeast  cells.  Bacteria  were  also  present  but  they  do 
not  show  in  the  mustration.—iH oward,  Yearbook  U.  S.  Dept.  of  Agriculture,  1911.) 

the  desirable  or  beneficent  forms  overgrow  the  objectionable 
forms  very  rapidly,  but  not  always.  It  should  be  one  of  the  prin- 
cipal efforts  of  the  food  and  drugs  bacteriologist  to  isolate  and 
identify  the  organisms  which  are  desirable  in  the  production  of 
fermented  food  products  and  those  which  are  unquestionably 
undesirable  and  objectionable,  for  in  these  products  it  is  not  a 
question  so  much  of  quantity  as  of  quality  of  the  organisms  present. 


56 


BACTERIOLOGICAL   METHODS 


This  is  by  no  means  a  simple  problem.  Much  of  this  field  of  work 
is  as  yet  untouched,  and  it  is  not  likely  that  definite  conclusions 
will  be  reached  in  the  very  near  future.  It  means  an  investigation 
of  those  conditions  which  are  recognized  as  diseases  in  industrial 
or  manufactured  products,  characterized  by  unaccountable  de- 


FiG.  1 6. — Mold  from  decomposing  plum, — {Howard,  Yearbook  U.  S.  Dept.  of 
Agriculture,  191 1.) 


teriorations  in  flavor,  in  taste,  in  color,  in  nutritive  value,  etc.  It 
means  a  very  careful  study  of  organisms  which  are  similar  in 
morphology  and  yet  quite  different  in  specific  functional  activities, 
giving  rise  to  objectionable  fermentation  products.  fl 

The  following  tables  will  give  some  idea  of  the  number  of  organ- 
isms which  occur  in  certain  canned  food  products.     Animal  foo( 


1 


DIRECT  EXAMINATION 


57 


products  are  not  included  in  Tables  II  and  III  because  there  are  not 
sufficient  data  available  on  which  to  base  suggestions.  There 
appears  to  be  no  plausible  reason  why  canned  animal  products 
should  not  be  subjected  to  the  same  method  of  examination  as 
vegetable  substances,  particularly  sausage  meats,  canned  meats, 
canned  oysters  and  shellfish  generally,  canned  eggs  and  canned 
soup  stocks.  Pickled  herring  which  shows  8,000,000,000  bacteria 
per  cc.  in  the  liquor  is  certainly  a  questionable  food  article.     In 


Fig.  17. — Spores  and  hyphal  fragments  from  decaying  sweet  pepper.     "Dry  rot" 
fungus. — {Howard,  Yearbook  U.  S.  Dept.  of  Agriculture^  191 1.) 

this  particular  instance  there  was  no  objectionable  odor  noticeable, 
but  the  meat  of  the  herring  was  somewhat  soft.  Smoked  meats 
and  fish  should  be  examined  for  mold  in  addition  to  bacteria. 
This  subject  should  receive  immediate  careful  consideration  on  the 
part  of  food  bacteriologists. 

Table  I  shows  the  number  of  organisms  which  may  occur  in 
some  of  the  more  common  household  food  substances,  fermented 
and  unfermented.  The  figures  are  based  upon  direct  counts. 
Table  II  is  based  upon  the  examination  of  factory  products  ob- 
tained in  the  open  market.  The  numerical  extremes  in  the 
micro-organisms  given  in  Table  II,  are  in  direct  ratio  to  the  relative 


S8 


BACTERIOLOGICAL   METHODS 
Table  I^' 


Number  of  Organisms  per  Cc, 

Hyphae 

Bacteria 

Yeasts 

Spores 

Blackberry  jam .  . 
Blackberry  jelly. . 
Cheese,  California 

Cider 

500,000 
Few        ... 

Few.                   j 

Few. 

80,000,000 

50,000  to 
500,000 
1,000,000 
Few 

Few 

Entirely       per- 
meated. 

50,000  to 
30,000,000 
Few 

Cider  vinegar. . .  . 

Currant  jelly 

Fruits,  canned 

Few 

Few 

Herring,  pickled. . 
Jams 

8,000,000,000 
Few 

Few- 

Few 

Few. 

Jellies 

Few 

Few 

Meat,  sausage 

Milk,  ordinary. . . 

Milk,  certified. . . . 

1,000,000  to 
150,000,000 
25,000  to 
2,000,000 
1,000  to 
15,000 
2,000,000,000  to 
7,000,000,000 
Few 

Milk,  sour 

i 

Plum  preserve... . 

Plum  relish 

Water,    drinking 
(San  F.). 

Few 

Few Few. 

100,000,000 

800  to 
32,000,000 

Few 

2,000,000      Some. 

t 

unsanitary  conditions  in  the  factories.  It  is  quite  evident  that 
the  products  of  the  manufacturers  who  employ  modern  methods  are 
fully  up  to  the  quality  of  those  prepared  by  the  careful  housewife. 

^  The  counts  recorded  in  Tables  I,  II,  and  III  were  made  by  the  direct  method 
using  the  hemacytometer.  In  the  case  of  the  sausage  meat  some  of  the  counts  were 
checked  by  the  plating  method  and  it  was  found  that  the  count  by  the  plating 
method  was  invariably  higher  than  by  the  direct  method.  Other  investigators  have 
noted  similar  discrepancies.  The  direct  examination  of  meats  for  bacteria  is  occasion- 
ally unsatisfactory  because  of  the  confusion  due  to  granular  fragments  traceable 
to  broken  up  blood  corpuscles,  fragments  of  coagulated  albumen,  etc. 


DIRECT  EXAMINATION 
Table  II 


59 


Number  of  Organisms  per  Cc. 


Name  of  Substance 


Bacteria 


Yeasts 


Spores 


HyphsB 


Apple  jam 

Apple  jam 

Apple  jam , 

Apricot  jam 

Blackberry   with 
apple. 

Catsup 

Catsup I    440,000,000 

Catsup I    560,000,000 

Catsup '    800,000,000 


500,000 

400,000 

25,000 

40,000 

5,000,000 


3,750,000 

494,000 

9,250,000 


Few. 


Some. 


Some 

i  None 

1,728,000        Numerous., 


Catsup 

Catsup 

Catsup. . . ;. 

Catsup 

Cherry  jam  with 
apple.  i 

Currant  jam  . ,  .  .  j 
Currant  jam  .... 

Fig  jam 1 

Loganberry  jam...i 

Loganberry  jam.,.' 

Orange     marma-  I 

lade  i 

Plum  jam ■ 

Peach  jam I 

Strawberry  jam.  . 
Strawberry  jam.  . 

Tomatoes 

Tomatoes 

Tomato  paste. .  .  . 
Tomato  paste. .  .  . 
Tomato  paste. .  .  . 
Tomato  paste. .  .  . 

Tomato  paste 

Tomato  paste.   .  . 


200,000,000 

5,000,000 

80,000,000 

400,000,000 
5,000,000 


12,000,000 


5,000,000 
27,500,000 
20,000,000 

1,500,000 
Few 


1,000,000 
Few 

2,500,000 


500,000 
1,250,000 


12,000,000 
2,000,000,000 
2,000,000,000 
2,000,000,000 
1,400,000,000 
4,000,000,000 
1,000,000,000 
2,000,000,000 


1,000,000 

40,000,000 

45,000 

1,250,000 

6,250,000 

8,750,000 


500,000 

750,000 

4,500,000 

500,000 


5,000,000 

7,500,000 

200,000 


Some. 
None. 
Very  abundant. 

Abundant. 
Very  abundant . 
Very  abundant. 
Entirely  per- 
meated. 
Very  abundant . 
Trace. 

Very  abundant. 
Very  abundant. 
Very  abundant. 


Some. 

Quite  abundant. 


10,000 


80,000,000 


Few 

750,000 
1,400,000 
4,000,000 
1,000,000 
1,200,000 
5,000,000 
6,000,000 
1,000,000 
100,000,000 


Some. 


Some. 
Abundant. 
Very  abundant. 
Very  abundant. 
Very  abundant. 
Very  abundant. 
Very  abundant. 
Very  abundant. 
Quite  abundant. 
Entirely  per- 
meated. 


6o 


BACTERIOLOGICAL  METHODS 
TABLE  II.— {Continued) 


Name  of  Substance 

Number  of  Organisms  per  Cc. 

Hyphae 

Bacteria 

Yeasts 

Spores 

Tomato  pulp^ 

Less  than 
5,000,000 

1,900,000,000 
Few 

Less  than 
500,000 

37,000,000 
Few 

Practically  none 
(1-3  per  cent. 

Tomato  pulp^. .  .  . 
Imitation  jam. . .  . 

of  fields). 
Entirely       per- 

30,000,000 

meated    (100 
per  cent.). 
Few. 

Table  III 


Name  of  Substance 


Maximum  No.  of  Organisms  per  Cc. 


Bacteria 


Yeasts 


Spores 


Hyphi 


Apple  butter 5, 000  to  1,000,000  to 

1,000,000  10,000,000 

Berries Few |  500,000 

Catsup 10,000,000  to  i  Few 

!      50,000,000       j 
500,000  to 
2,000,000 
Few 


500,000    '  15  per  cent. 


Cider. 
Fruits. 
Jams. . 


500,000 


18  per  cent. 


1,000,000 
Few 


500,000  to 
5,000,000 

50,000  to  j       500,000  tO:  10  to  1 2  per  cent. 
500,000       j    1,000,000 
1,000,000  to        500,000 
10,000,000 
1,000,000         Few 


Jellies 

Marmalade^ | 

Tomato  pastes. .  .     500,000,000       |  Few 2,000,000 


Vinegars  (fruit) 


5,000,000 


Few. 


10  per  cent. 
I  to  5  per  cent. 


20     to    25    per 
cent.  -M 


1  Both  samples  were  from  large  factories  and  represent  the  extremes  in  the  factor 
conditions.     The  first  sample  is  from  a  factory  where  the  conditions  are  what  th 
should  be,  the  second  from  a  factory  where  the  conditions  are  just  the  reverse. 

2  Percentages  given  this  column  refer  to  the  number  of  the  1/125  c.mm.  areas 
the  mold  counter  described  in  Fig.  5  which  contain  hyphal  clusters.     As  a  rule  abu: 
dant  spores  indicate  the  presence  of  abundant  hyphal  tissue,  and  vice  versa. 

^  The  organism  in  orange  marmalade,  under  ordinary  conditions  of  manufacture, 
are  negligible  in  amount. 


1 


DIRECT   EXAMINATION  6 1 

Other  manufacturers,  either  through  greed,  ignorance  or  careless- 
ness, or  through  all  three  causes  combined,  refuse  to  employ- 
modern  methods  and  as  a  result  their  products  are  very  often  in 
an  undescribably  filthy  condition,  wholly  unfit  for  consumption. 
In  addition  to  the  bacteria,  yeast  cells,  mold,  sand  and  dirt 
particles  present  in  the  inferior  grades  of  catsup,  jams,  jellies, 
etc.,  there  are  found  insect  remnants  (flies,  aphides,  beetles), 
vinegar  eels,  larvae  of  various  nematodes  (from  soil),  etc.  The 
presence  of  numerous  fly  remnants  is  certainly  an  indication  of 
highly  unsanitary  factory  conditions.  The  presence  of  vinegar 
eels  indicates  the  use  of  bad  vinegar  and  the  presence  of  soil 
nematodes  and  of  sand  and  dirt  particles  indicates  insufficient  or 
no  washing.  Laboratory  experience  has  demonstrated  that 
there  is  a  definite  relationship  between  the  number  of  bacteria 
and  other  organisms  and  the  amount  of  dirt  and  other  impurities 
present  in  factory  products.  Unsanitary  factory  conditions  en- 
courage a  certain  recklessness  in  such  factories,  inducing  the 
laborers  about  the  place  to  even  go  out  of  the  way  to  add  more 
filth.  Thus  shovelfuls  of  refuse  are  taken  up  from  the  filth-coated 
floors  and  thrown  into  the  mixing  vats,  the  idea  evidently  being 
that  it  will  add  to  the  bulk  and  that  no  one  will  know  the  difference. 
Vats  are  often  not  cleaned  until  the  conditions  are  almost  unde- 
scribable.  Refuse  is  added,  often  of  such  a  character  as  to  be  un- 
fit as  food  even  for  animals.  This  criminal  negligence,  care- 
lessness and  indifference  is  too  frequently  engendered  by  ignor- 
ance which,  gives  heed  to  nothing  else  than  a  strict  enforcement 
of  the  law. 

The  filthy  condition  of  some  of  these  products  is  very  generally 
not  apparent  to  the  layman  because  of  certain  methods  employed 
primarily  intended  to  hide  or  mask  such  defects.  The  odors  of 
decomposition  are  quite  effectually  dissipated  by  the  steaming 
and  cooking  process.  The  vitiated  taste  is  quite  effectually 
masked  by  the  heavy  spicing.     Any  appreciable  change  in  color  is 


62  BACTERIOLOGICAL   METHODS 

restored  by  means  of  added  coloring  substances.  Any  change 
in  consistency  is  corrected  by  adding  fillers,  such  as  starch,  gelatin 
and  agar.  The  unscrupulous  manufacturer  will  work  up  a  supply 
of  spoilt  canning  tomatoes,  including  rejected  ''swells"  and 
*'leaks,"  making  them  into  catsup  or  paste.  Overripe  and  par- 
tiany  decomposed  fruits  (culls  and  rejects)  are  worked  up  into 
jams  preserves  and  into  combinations  in  which  the  objectionable 
character  and  appearance  are  hidden  or  lost  sight  of. 

We  are  justified  in  the  conclusion  that  the  number  of  micro- 
organisms in  food  products  is  a  reliable  guide  to  the  wholesomeness 
and  sanitary  quality  of  such  products  and  the  very  natural  ques- 
tion arises,  what  are  the  maximum  numbers  of  bacteria,  yeast  cells 
and  mold  spores  (including  mold  hyphae)  permissible  under 
reasonable  and  practicable  sanitary  -conditions.  While  ideal 
factory  conditions  may  not  always  be  practicably  attainable, 
yet  it  is  wholly  reasonable  to  expect  the  operation  or  methods  which 
will  bring  the  maximum  quantitative  counts  per  cc.  within  the 
numerical  limits  given  in  Table  III.  These  proposed  maximum 
numerical  limits  are  tentative  only.  As  the  sanitary  conditions 
in  the  canneries  are  improved,  as  they  undoubtedly  will  be,  the 
limits  can  be  correspondingly  decreased,  finally  reaching  the 
negligible  quantities  as  already  explained.  Where  numbers  are 
omitted  in  the  tables  it  indicates  that  the  quantity  of  organisms 
is  negligible.  "Few,"  indicates  that  the  number  of  organisms  is 
somewhat  more  than  in  negligible  amounts,  yet  not  sufficient  to 
make  counting  necessary  or  to  question  the  suitableness  of  the 
article  for  food  purposes. 

It  is  quite  evident  that  different  numerical  limits  must  be 
adopted  for  different  classes  or  kinds  of  food  products.  This  can 
be  seen  from  a  study  of  the  tables.  Some  fruits  and  fruit  products 
are  more  susceptible  to  the  attacks  by  bacteria,  yeasts  and  mold^B 
than  others.  Acid  fruits,  as  the  cherry,  the  plum,  tomatoes, 
loganberries,  blackberries,  etc.,  are  much  more  likely  to  be  attackec 


icked^ 


DIRECT   EXAMINATION  63 

by  molds  than  are  apples,  peaches,  pears  and  apricots.  Yeasts 
very  rarely  appear  in  the  whole  fruit,  but  they  develop  very 
rapidly  in  fruit  pulps  which  contain  sugar  (natural  or  added). 
Yeasts  require  in  addition  to  sugar,  a  high  percentage  of  moisture 
for  their  active  growth,  including  an  ample  supply  of  oxygen  (air) . 
The  presence  in  canned  fruit  products  of  numerous  yeast  cells 
indicates  fermentation  during  the  processing.  The  presence  of 
numerous  bacteria  in  fruit  products  indicates  the  use  of  rotted 
(bacterially)  fruit  or  bacterial  contamination  and  development 
during  the  processing,  or  both. 

It  would  appear  that  most  of  the  bacteria  which  develop  in  fruit 
pulps,  especially  those  from  fruits  which  are  quite  acid,  as  for  ex- 
ample tomato  pulps,  belong  to  the  lactic  acid  group.  Numerous 
tests  in  the  laboratories  of  the  Bureau  of  Chemistry  show  a  paral- 
lelism between  the  number  of  bacteria  and  the  amount  or  per- 
centage of  lactic  acid  present  in  tomato  catsups.  The  usual 
rotting  bacteria  require  more  air  (oxygen)  then  is  present  in  the 
pulp  mass  and  as  a  result  these  are  soon  overgrown  by  the  lactic 
acid  bacilli,  if  the  pulp  is  allowed  to  stand  for  a  time  without  steril- 
ization. It  is,  however,  very  evident  that  the  contamination  of 
such  products  as  catsups,  tomato  pastes  and  tomato  purees  is 
never  wholly  limited  to  lactic  acid  bacilli.  The  inclusion  of  field 
rotted  tomatoes  and  the  rotted  pulp  material  from  filthy  mixing 
vats  and  other  parts  of  the  machinery  of  the  unsanitary  factories, 
adds  a  sufficient  number  of  rotting  bacteria  to  render  the  article 
dangerous  to  health,  if  consumed.  Ravenel  and  other  investiga- 
tors have  shown  that  when  certain  food  products,  as  cream  and 
milk,  are  kept  in  cold  storage,  particularly  after  pasteurization 
or  incomplete  sterilization,  the  development  of  lactic  acid  bacilli 
is  checked  and  the  growth  of  toxin  forming  bacteria  is  encouraged, 
resulting  in  occasional  poisoning ,  to  the  consumer.  It  is  very 
likely  that  similar  conditions  may  exist  in  some  of  the  incompletely 
sterilized  canned  food  products  (vegetable  as  well  as  animal)  which 
have  been  stored  for  some  time  at  a  comparatively  low  temperature. 


64  BACTERIOLOGICAL    METHODS 

The  question  is  frequently  asked,  what  percentage  of  rotten  or 
moldy  fruit  must  be  present  to  render  the  product  unfit  for  human 
consumption?  This  question  cannot  be  answered  definitely.  In 
a  general  way,  it  may  be  stated  that  where  there  is  not  over  5  per 
cent,  of  rotted  or  moldy  fruit  used,  the  number  of  organisms  in  the 
finished  products  will  not  reach  the  maximum  limits  given  in  Table 


Fig.  18. — A  type  of  mold,  Spicaria  sp.,  very  frequently  found  on  decaying 
tomatoes.  Some  of  the  filaments  and  numbers  of  spores  are  shown. — {Howard, 
Yearbook  U.  S.  DepL  of  Agriculture,  191 1.) 

Ill,  in  fact  the  counts  will  in  all  probability  be  considerably  less. 
A  careful  culling  of  spoilt  fruit  in  the  field  and  at  the  factory, 
coupled  with  reasonably  sanitary  factory  methods  and  modern 
methods  of  sterilization,  will  furnish  products  which  will  meet  all 
of  the  requirements  of  any  pure  food  law. 

The  statement  is  frequently  made  by  manufacturers  that  even 


DIRECT  EXAMINATION  65 

though  bacteria,  yeasts  and  mold  are  present  in  considerable 
numbers,  they  are  harmless  and  do  not  produce  toxic  effects 
when  introduced  into  the  digestive  tract.  This  statement  is 
wholly  without  foundation  in  fact.  On  the  contrary  it  is  known 
that  certain  bacteria,  yeasts  and  molds  do  cause  disease  and 
more  or  less  severe  intoxications  and  intestinal  disturbances. 
The  objectionable  character  of  mold  is  universally  recognized 


Fig.  19.— Mold  colonies  in  gelaiiii  o^cn  uini^i  liic  luw  pu>\ci  ot  the  microscope 
(X  80).  This  mold  developed  in  the  gelatin  after  it  was  spread  on  the  screen 
to  dry.  This  gelatin  also  contained  numerous  bacteria.  Gelatin  thus  infected  is 
not  suitable  for  bacteriological  purposes  neither  is  it  suitable  for  use  as  food. 

and  nearly  all  animals  refuse  to  eat  moldy  and  mold  contaminated 
food  materials.  Various  ulcerative  diseases  of  the  skin  and  of 
the  digestive  tract  are  caused  by  mold  organisms.  While  many 
of  the  yeasts  are  entirely  harmless  and  cause  very  important 
fermentative  changes,  some  of  them  are  pathogenic  to  man  while 
others  initiate  objectionable  fermentation  changes  in  the  food 
substances. 


66 


BACTERIOLOGICAL  METHODS 


As  already  indicated  the  number  of  organisms  in  food  sub- 
stances is  in  direct  ratio  to  the  following  conditions : 

1.  Insufficient  culling  of  partially  and  wholly  decomposed  fruits. 

2.  Unsanitary  factory  conditions  and  unsuitable  methods. 


Ova  Of  the  Parasitic  Worms  or  Man 
TREMATODA 


DRAWN  TO  8    C    A    L    C  X       leOO 


Heterophyes 
heterophyes 

(o.fU:rLooas.igos)i 


/  2 


,        ,     ,     Opisthorchis 

i(     :l^K^l  '"'■■  I  fclineUS (».f«crLooaa.KJ06) 


Dicro-  ,, 

coeliumVs 

loinceatum 

^nr...o.. ...;     Fasclola  Texsciolopsis 


Clonorchis  Clonorchis 
sinensis  endemicus 


SchistoV       Schistosoma  pcragoninius  ^  ^^V  ?^?,\\'k  o  J^^^' 
soma  V  japonicum  ^  westeinnanii       ff^S,^^^  Schistosoma    ^ 


U.SAaia/  Metlioa/ Sc/}OQl. 


Fig.  2o. — Intestinal  ova.  Trematodes.  Ova  of  intestinal  parasites  may  possibly 
occur  in  foods  of  vegetable  origin  contaminated  by  soil,  sewage  and  fecal  matter. 
Note  comparative  size  and  the  actual  measurements  according  to  the  scale.  It  may 
be  mentioned  that  the  extremely  small  seeds  of  Vanilla  planifolia  have  been  mistaken 
for  ova  of  intestinal  parasites. — {Stitt.) 


We  are  warranted  in  establishing  a  maximum  limit  as  to  the 
number  of  organisms  permissible  in  food  substances.  The 
method  of  estimating  the  quality  of  foods  based  upon  the  number 
of  micro-organisms  present  has  been  tested  out  in  different  coun- 
tries and  has  proven  very  reliable  and  satisfactory;  and  those  who. 


DIRECT   EXAMINATION 


67 


■die  entrusted  with  the  enforcement  of  the  laws  governing  the 
physical  well-being  of  the  people  are  most  emphatically  in  the 
right  when  they  insist  that  the  sanitation  in  and  about  our 
factories  should  be  of  a  high  order. 

In  addition  to  the  purely  quantitative  estimates  of  micro- 
organisms based  upon  direct  examination,  the  analyst  is  enabled 


Ova  or  the  Parasitic  Worms  or  Man 
CESTODA 

ORAVVfM  TO  SCALE  X  lOOO 


(....  .o.«,M.)      ^onoporus    cephalus       Hymenolepis  ne^na 

§^gmndis       latus(.ree..oo«,„«)         (.a.rR....n.. «,...,..  ,^.) 
jtj^  \  diminuta 


Teenia 
sadinata^ 


«  Rafter Bi2.-oirri.  f-cniS(i!rj  K)00j 


Dipylidiuiii 
caninum(nf,...,u,.uw.) 

Costode  sepments 

'O    SCALE    X   10       W 


10  MtLLIMlTCRS,        ___ 

■    '    "    1,1    I    !    I    I      -^ 


# ,  i    Davainea  __ 

lepls 
a^T        diminuta,. 


Tbenia  Dipylidium  ^^*-^^.;r^zj^-.    -  j^  nana'^S^^^  4 

solium  caninum    Dibothriocephalus  latus  saginata 

iMea/'f  US-A/nya/MedlcaJSc/iOQl. 


Fig.  2 1. -^Intestinal  ova.     Cestodes. — {Stitt^i 


to  form  certain  opinions  and  conclusions  regarding  the  source  of 
the  contamination.  For  example,  the  hyphal  development  in 
mold  infested  fruit  is  in  marked  contrast  to  the  hyphal  develop- 
ment in  the  fruit  pulp,  due  to  unsanitary  factory  conditions. 
This  difference  in  hyphal  structure  is  due  to  a  difference  in  the 
amount  of  oxygen  (air)  supply,  of  moisture,  of  light  and  the 
6 


68  BACTERIOLOGICAL  METHODS 

added  ingredients  (spices,  sugar,  vinegar,  etc.),  of  the  canning 
product.  The  analyst  can  thus  determine  approximately  how 
much  of  the  hyphal  tissue  present  is  derived  from  the  use  of 
moldy  fruit  and  how  much  is  traceable  to  unsanitary  factory 
conditions.  Again,  the  presence  of  one  or  more  ova  or  larvae  of 
intestinal  parasites,  as  the  tape  worm,  would  indicate  sewage 
contamination  or  contamination  with  fecal  matter.  Sand  and 
dirt  particles  indicate  insufficient  washing,  etc.  It  is  self  evident 
that  the  value  of  the  report  by  the  analyst  depends  upon  his 
knowledge  of  the  subject  and  the  range  of  his  experience.  Until 
the  work  is  well  under  way  and  the  methods  are  perfected,  there 
is  no  place  for  inexperienced  analysts  in  our  food  and  drugs 
laboratories. 

4.  Quantitative   Estimations   by   the    Cultural   Methods 

Estimating  the  number  of  bacteria  per  cc.  in  foods  and  drugs, 
etc.,  by  planting  or  plating  definite  amounts  of  the  substances 
into  plate  (Petri  dish)  culture  media,  is  a  well-known  and  standard 
procedure.  The  general  and  special  technique  of  the  plating 
method  is  described  in  the  various  text-books  and  manuals  on 
bacteriology.  Some  of  the  details  of  the  method  are  standard, 
in  so  far  as  they  are  generally  adopted  by  investigators,  such 
as  the  preparation  of  certain  culture  media,  making  the  dilutions, 
counting,  etc.;  in  other  regards  there  is  anything  but  uniformity. 
It  is  generally  admitted  that  the  results  of  different  investigators 
differ  widely  but  there  appears  little  unanimity  of  opinion  as  to 
the  factors  which  are  responsible  for  these  variations  in  quanti- 
tative results. 

Micro-organisms  are  sensitive  to  a  degree  and  they  respond 
readily  to  the  slightest  variations  in  moisture,  temperature  and 
food  supply.  A  failure  to  recognize  this  fully  in  laboratory 
practice  leads  to  confusion  and  erroneous  results.  The  following 
are  some  of  the  more  important  factors  which  are  responsible  for 
errors  and  variations  in  results. 


I 


CULTURE   MEDIA 


69 


I.  Culture  Media. — Dififerences  in  the  quality  of  the  meat 
used  in  making  the  meat  infusions  has  given  some  marked  varia- 
tions in  the  quantitative  results.  Meat  extracts  from  younger 
animals  give  higher  counts  than  do  extracts  from  the  meat  of 
older  animals.  Again,  the  prepared  extracts  of  the  different 
packing  houses  give  different  results  and  the  results  obtained 


Ova  of  the  Paroisitic  Worms  or  Man 
NEMATODA 


O  R  AW 


A- Median  focus    BrSurfoice  focus 


/Modijicd  from  Stiles J<WI.\      ( ».fter  Stiles.  19M\ 
\aria  Looss  1405  /       ^  / 


tnetricexUy  it  >    '  j 
convex  ['   '    ] 

(a  fter  looit.HfS^^Jj 

Oxyuris     , 

vermlcularisA.B 


show  one 
s^fUvtten- 


U.-Alypicix.l.  urrfertilized 

CQQ    ((\ftorLooss.l9W) 


Strongylus 
subtilis 

(ftfter  ioosv  1905J 


Cr vslthout  outer 
envelope  (Modir.ed  > 

iroa  Sfi!es,lVti2.and       \ 


Loois,l90S^ 


,.  ,_y  A0chyloslomoi 
S^^ew*;^.  duodena le 


Asca.ris 
lumbricoides 
A. BCD.        Trichuri.s    '''°«"'w 
tnchiurex 

lOrigiiiAl) 


Necevtor 
americanus 


lMi;>.^ws....,>,o..M   ^f^sftooi 


Embryo 
in  stool 
after  i2  to48 

Agchylostomol 
duodenale 


Strongyloides 
stercorcdis  , 

i/SAaya/.Wed/caJSc/iOQ/. 


Fig.  22. — Intestinal  ova.     Nematodes. — i^Stitt.) 


from  the  use  of  media  made  with  manufactured  meat  extracts 
differ  from  those  obtained  from  the  use  of  the  laboratory  made 
meat  infusions.  In  fact  most  workers  are  opposed  to  the  use  of 
the  manufactured  meat  extracts  because  of  the  fact  that  they  are 
mixed  products  and  also  because  of  the  uncertainty  of  the  amount 
and  number  of  the  added  ingredients  any  or  all  of  which  may 


70  BACTERIOLOGICAL  METHODS 

interfere  with  the  growth  and  development  of  certain  bacteria. 
Investigators  have  also  noted  great  variations  in  results  with 
different  brands  or  makes  of  peptones  used,  the  quantitative 
differences  amounting  to  50  per  cent,  in  some  cases.  Equally 
remarkable  are  the  differences  due  to  the  kinds  of  water  used  in 
the  preparation  of  the  culture  media.  For  instance  it  is  known 
that  agar  made  up  with  sewage  encourages  the  development  of 
sewage  organisms  while  the  same  medium  made  up  with  tap 
water  encourages  the  growth  of  bacteria  predominating  in  such 
tap  water. 

The  gelatine  used  is  yet  another  important  factor  in  cultural 
results,  depending  upon  the  age  of  the  gelatine,  its  purity,  the  de- 
gree of  heating  to  which  it  has  been  exposed,  its  origin,  possible 
contamination  with  arsenic,  with  bacteria  and  mold.  Other 
ingredients  used  in  the  preparations  of  culture  media  cause  more 
or  less  marked  variation  in  comparable  results.  The  above 
statements  make  it  evident  that  it  is  absolutely  necessary  to 
adopt  and  to  adhere  to  uniform  methods  in  order  that  the  com- 
parable results  may  be  approximately  uniform. 

2.  Glassware. — Different  investigators  have  found  that  the 
number  of  bacteria  in  and  upon  culture  media  varied  with  the 
composition  of  the  glass  containers  used.  The  comparatively 
soluble  glass,  for  example,  yielded  enough  free  alkali  to  inhibit  the 
development  of  the  more  sensitive  bacteria.  The  size  of  the 
containers  and  the  thickness  of  the  glass  yielded  differences  in 
the  results.  It  is  therefore  very  desirable  to  adopt  Petri  dishes 
and  test-tubes  of  standard  form  and  thickness  of  standard  cubic 
contents. 

3.  Other  Factors. — The  form  and  size  of  the  incubating 
chamber,  the  degree  of  ventilation,  degree  of  darkness,  amount  of 
oxygen  present,  etc.,  cause  variations  in  the  results. 

Of  even  greater  importance  than  any  of  the  factors  so  far 
mentioned,  is  the  personal  equation  in  the  laboratory  technique. 
No  two  workers  follow  out  the  same  details  in  the  different  steps 


CULTURE    MEDIA  7 1 

of  the  laboratory  procedure  and  very  frequently  proper  judgment 
is  lacking  in  the  application  of  certain  details  of  the  methods. 
For  example,  there  is  lack  of  uniformity  in  the  degree  of  heat 
to  be  used  in  melting  gelatin  media  preparatory  to  planting, 
in  the  amount  of  material  to  be  planted  in  each  Petri  dish,  manner 
of  planting,  time  of  incubating,  etc. 

We  hereby  submit  the  following  technique  in  the  preparation 
of  culture  media  and  in  the  methods  of  making  cultures  in  plates 
as  well  as  in  test-tubes,  following  very  generally  the  suggestions 
as  given  in  the  report  of  the  Committee  of  the  American  Health 
Association. 

5.  Preparation  of  Standard  Cultural  Media,  General  Suggestions 

I.  Ingredients. — Distilled  water  is  to  be  used  in  the  prepara- 
tion of  all  of  the  standard  media.  The  distilled  water  must  be 
comparatively  free  from  bacteria  and  must  be  kept  in  clean 
sterilized  containers  and  as  free  as  possible  from  mineral  and 
organic  impurities.  If  other  than  distilled  water  is  used,  this  is 
to  be  stated  and  the  special  reasons  for  using  it  indicated. 

For  making  meat  infusions,  fresh  lean  meat  is  to  be  used, 
from  comparatively  young  animals,  free  from  disease.  Meat 
extracts  may  be  used  in  place  of  the  meat  infusion. 

Unless  otherwise  specified,  the  peptone  used  should  be  made 
from  fresh  beef  by  pancreatic  digestion.  It  should  be  dry  and 
recently  made.  Workers  should  be  sure  to  specify  the  kind  of 
peptone  desired.  Egg  albumen  or  fibrin  peptone  is  not  to  be 
used  in  any  of  the  standard  media.  The  article  should  be  secured 
from  some  reliable  house. 

The  gelatin  to  be  used  in  the  preparation  of  the  standard  media 
should  be  of  the  best  obtainable,  the  so-called  French  brand  be- 
ing, as  a  rule  preferred.  A  lo  per  cent,  solution  should  not 
soften  when  kept  at  a  temperature  of  25°  C.  It  should  be  en- 
tirely free  from  arsenic  and  as  free  as  possible  from  acids,  micro- 
organisms, molds,  and  other  impurities.     A  good  grade  of  gelatin 


72  •  BACTERIOLOGICAL  METHODS 

should  respond  to  the  following  test:  Place  0.30  gram  of  the 
gelatin  in  a  medium  sized  test-tube  and  add  15  cc.  of  distilled 
water,  let  stand  for  half  an  hour,  warm  gently  until  all  of  the 
gelatin  has  dissolved,  then  place  the  tube  in  water  at  a  tempera- 
ture of  15.5°  C.  and  leave  undisturbed  for  half  an  hour.  The 
solution  should  remain  in  place  when  the  tube  is  inverted. 

The  commercial  gelatin  is  a  variable  product,  being  made  from 
varying  proportions  of  animal  tissues  as  hides,  ligaments,  bone  and 
bone  cartilage.  The  piirest  and  best  gelatin  is  made  from  liga- 
ments and  this  kind  would  no  doubt  give  the  most  uniform  re- 
sults in  bacteriological  work,  but  it  is  apparently  not  possible 
to  obtain  such  gelatin  in  the  market.  The  next  best  grade  (prac- 
tically obtainable)  would  be  that  made  from  hides  of  compara- 
tively young  domestic  cows  free  from  all  foreign  additions  as  salt, 
arsenic  and  other  hide  preservatives. 

Each  lot  of  gelatin  should  be  examined  microscopically  before 
making  it  into  culture  media.  Old  yellowed  and  brittle  material 
should  not  be  used.  Examine  from  five  to  six  sheets  from  each 
pound  package,  using  the  low  power  of  the  compound  microscope. 
The  examinations  are  made  directly  without  mounting.  If  numer- 
ous mold  colonies  are  found  as  shown  in  Fig.  19,  or  numerous 
mold  filaments  more  or  less  scattered  through  the  mass,  it  is 
unfit  for  use  as  a  culture  medium.  Numerous  formed  mold 
colonies  in  the  matrix  indicate  growth  during  the  drying  process 
after  the  gelatin  was  spread  on  the  drying  screens.  More  or 
less  torn  and  disintegrated  hyphal  fragments  unequally  distributed 
through  the  mass  indicate  infection  and  growth  before  the 
gelatin  was  spread  for  drying.  To  examine  for  bacteria,  mount 
small  bits  of  the  sheet  on  a  slide  in  water  covered  with  cover 
glass.  If  bacteria  are  numerous,  approximating  10,000,000  per 
cc.  and  more,  it  should  not  be  used.  In  order  to  make  more 
accurate  counts,  take  i  gram  of  the  gelatin  and  rub  up  in  9  cc. 
of  boiled  distilled  water  and  make  the  counts  of  the  thoroughly 
mixed  sample  by  means  of  the  hemacytometer.     As  a  rule  it  is 


STERILIZATION  73 

not  necessary  to  make  plate  or  tube  cultures  to  determine  the 
fitness  of  the  gelatin  for  bacteriological  work.  Incidentally  it 
may  be  remarked  that  a  gelatin  which  is  unsuitable  for  bacterio- 
logical work  is  also  unfit  for  use  as  human  food. 

The  agar  should  be  the  highest  grade  obtainable,  and  if  the 
shredded  form  is  used  it  should  always  be  washed  in  sterilized 
distilled  water  before  making  into  culture  media. 

With  regard  to  the  other  ingredients  required  in  making  cul- 
ture media,  such  as  dextrose,  lactose,  maltose,  saccharose,  glycerin, 
salt  litmus,  etc.,  etc.,  special  efforts  should  be  made  to  get  these 
as  pure  as  possible.  The  degree  of  purity  should  be  determined 
by  actual  tests. 

2.  Sterilization. — Thorough  sterilization  of  all  culture  media 
is  absolutely  necessary.  It  is,  however,  known  that  heating  pro- 
duces some  marked  changes  in  the  molecular  composition  of  the 
media,  even  inducing  actual  chemical  decomposition.  It  is 
therefore  desirable  to  make  the  time  of  heat  exposure  as  brief  as 
possible.  Ordinarily  it  is  therefore  preferable  to  use  the  auto- 
clave, bringing  the  temperature  up  to  120°  C.  (15  lb.  pressure) 
for  a  period  of  15  min.  This  temperature  will  sterilize  all 
media.  A  shorter  period  does  not  insure  complete  sterilization 
and  a  longer  exposure  is  apt  to  produce  inversion  of  the  sugars 
used  and  also  permanently  lower  the  melting  point  of  the  gelatin. 
Solid  media  as  gelatin  and  agar  should  be  liquefied  before  placing 
in  the  autoclave. 

The  following  rules  should  be  strictly  observed  in  using  the 
autoclave : 

a.  The  sterilizer  should  be  hot  when  the  media  are  introduced. 
About  100°  C.    Let  all  air  escape  from  chamber. 

b.  At  the  end  of  the  period  of  sterilization  (15  min.),  remove 
the  media  and  cool  them  as  rapidly  as  possible. 

Compliance  with  these  rules  will  reduce  to  a  minimum  the 
tendency  toward  liquefaction  of  the  gelatin  and  a  tendency  to 
decompose  the  various  chemicals  used,  due  to  prolonged  heating. 


74  BACTERIOLOGICAL   METHODS 

If  streaming  or  live  steam  is  to  be  used  in  place  of  the  steam 
under  pressure  in  the  autoclave,  intermittent  sterilization  is  to 
be  practised.  Place  the  media  in  the  steam  sterilizer  for  30 
min.  on  each  of  3  successive  days.  Wait  until  the  tem- 
perature in  the  sterilizer  has  risen  to  approximately  100°  C. 
before  placing  the  media  therein.  Agar  media  should  first  be 
liquefied.  At  the  end  of  each  period,  remove  the  media  and  cool 
as  rapidly  as  possible  for  reasons  already  given. 

When  media  are  prepared  under  the  proper  laboratory  condi- 
tions and  sterilized  as  above  suggested,  they  are  as  a  rule  free  from 
all  living  germs.  However,  if  practicable,  the  media  should  be 
watched  for  a  period  of  2  days,  stored  in  a  room  at  ordinary 
temperature,  in  order  to  note  possible  bacterial  developments. 

3.  Adjustment  of  Reaction  of  the  Media. — As  a  rule  bacteria 
develop  most  actively  in  media  which  are  slightly  alkaline  to 
litmus  and  since  certain  media  are  quite  acid  in  reaction  (gelatin 
in  particular)  it  becomes  necessary  to  reduce  them  to  a  standard 
reaction.  The  standard  indicator  to  be  used  is  phenolphthalein. 
When  phenolphthalein  is  not  obtainable,  litmus  paper  (or  a  i 
per  cent,  aqueous  solution  of  Kahlbaum's  azolitmin)  may  be 
used.     The  reaction  adjustments  are  to  be  made  as  follows: 

Place  5  cc.  of  the  medium  to  be  tested  in  45  cc.  of  distilled 
water  (making  a  dilution  of  i-io).  Boil  briskly  for  i  min., 
with  stirring  or  rotary  shaking.  Add  i  cc.  of  the  phenolphthalein 
solution  (made  by  dissolving  5  grams  of  the  salt  in  i  liter  of 
50  per  cent,  alcohol).  Titrate  while  hot  with  N/20  caustic 
soda  solution  (in  distilled  water).  A  distinct  pink  coloration 
marks  the  proper  reaction.  To  be  more  precise,  the  pink  should 
correspond  to  a  mixture  or  combination  of  25  per  cent,  red  and 

75  per  cent,  white  of  the  color  top  recommended  by  the  Com- 
mittee on  Standard  Methods  of  the  American  Health  Associa- 
tion. The  reactions  of  the  media  are  stated  in  terms  of  the 
percentages  of  normal  acid  or  alkaline  solutions  required  to 
neutralize    them.     Alkalinity   is    indicated    by  the  minus  (  —  ) 


STANDARD   MEDIA  75 

sign  and  acidity  by  the  plus  (  +  )  sign.  Thus,  if  the  reaction 
of  a  medium  is  given  as  +  i-oo  it  indicates  that  it  would  be 
necessary  to  add  i  per  cent,  of  normal  sodic  hydrate  solution  to 
the  medium  in  order  to  bring  it  to  the  neutral  point  (to  phenol- 
phthalein).  It  will  be  observed  that  while  the  titrating  is  done 
with  the  N/20  caustic  soda  solution,  the  normal  solution  is  added 
to  bring  the  medium  to  the  desired  reaction,  the  stronger  solution 
being  preferred  because  it  reduces  the  amount  of  liquid  intro- 
duced. The  Committee  on  Standard  Methods  specifies  that  the 
reaction  of  all  standard  culture  media  shall  be  +  i-o  per  cent, 
and  if  it  differs  in  reaction  by  more  than  0.20  per  cent,  the  medium 
shall  be  readjusted  and  when  a  reaction  other  than  the  standard 
is  used  it  shall  be  indicated  and  the  reasons  for  using  a  different 
reaction  shall  be  fully  stated. 

Media  are  preferably  made  in  large  quantities  as  this  will 
reduce  to  a  minimum  the  discrepancies  due  to  variation  in  the 
composition  of  the  ingredients  used.  As  soon  as  made  and 
titrated,  the  media  should  be  put  into  tubes  and  in  other  culture 
containers,  after  which  media  containers  and  all  are  to  be  sterilized 
according  to  the  methods  already  described.  To  guard  against 
the  evaporation  of  moisture  from  the  media,  the  tubes,  flasks,  etc., 
should  be  sealed  by  dipping  the  plugged  ends  into  melted  paraffin, 
or  they  may  be  capped  with  rubber  coverings  especially  made 
for  that  purpose.  In  case  media  are  to  be  used  within  a  few 
days,  sealing  is  not  necessary  but  they  should  be  kept  in  a  moist 
place,  preferably  in  the  ice-box. 

6.  Preparation  of  Required  Standard  Culture  Media 

Culture  media  used  in  bacteriological  work  may  be  divided 
into*  those  which  are  required  for  general  purposes  and  those 
which  have  special  uses.  The  former  should  by  all  means  be 
prepared  according  to  the  standards  suggested  by  the  Committee 
of  the  A.  H.  A.  If  special  media  are  used,  their  exact  composi- 
tion and  mode  of  preparation  should  be  fully  and  explicitly  given. 


76  BACTERIOLOGICAL   METHODS 

Furthermore,  the  reasons  why  the  special  media  are  used  should 
be  clearly  set  forth,  so  that  co-workers  may  judge  of  their  special 
value  and  may  try  them  out  intelH gently,  should  they  care  to 
do  so. 

As  special  media  are  adopted  into  general  use  by  the  majority 
of  bacteriologists  they  are  to  be  relegated  into  the  group  of  general 
media.  For  example,  a  few  years  ago,  lactose-litmus-agar,  Endo 
medium,  Hess'  medium,  lactose-bile  medium,  etc.,  were  special 
media.  They  are  now  in  general  use  and  they  should  be  pre- 
pared according  to  a  standard  method.  We  hereby  give  the 
methods  of  preparing  some  of  the  more  important  media  used  in 
general  bacteriological  work,  following  the  directions  of  the 
Committee  of  the  A.  H.  A. 

I.  Nutrient  Broth. — Infuse  500  grams  of  chopped  lean  meat 
for  24  hr.  in  distilled  water.  Shake  occasionally  and  keep  in 
the  refrigerator.  Any  loss  by  evaporation  is  to  be  restored. 
Strain  the  infusion  through  cotton  or  through  cotton  flannel. 
Add  I  per  cent,  of  peptone  and  warm  over  water  bath  or  steam 
until  the  peptone  is  entirely  dissolved.  Heat  for  30  min.  in 
rice  cooker  or  in  steam  sterilizer  and  restore  any  loss  by  evapora- 
tion. Titrate  with  normal  sodic  hydrate  (or  normal  hydro- 
chloric acid)  to  a  reaction  of  +  i-o  per  cent.  Boil  for  2  min. 
over  open  flame,  stirring  constantly.  Restore  loss  by  evapora- 
tion. Filter  through  cotton  (placing  the  cotton  on  cotton 
flannel  or  on  perforated  filter  paper).  Pass  the  medium  through 
this  filter  until  it  comes  out  perfectly  clear.  Again  titrate  and 
record  the  final  reaction.  Pour  into  tubes  (10  cc.  in  each  tube) 
and  sterilize  in  the  manner  as  already  directed. 

This  medium  is  much  used  for  general  cultural  purposes. 
It  is  used  in  making  the  cultures  of  typhoid  fever  germs,  for 
determining  the  phenol  coefficient  of  disinfectants  by  the  Ander- 
son-McClintic  method  of  rating  the  germ  destroying  power  of 
disinfectants.  It  is  also  used  in  culturing  motile  bacteria,  etc. 
Variousjndicators  may  be  added. 


\  STANDARD  MEDIA  77 

2.  Sugar  Broths. — Broths  to  which  sugars  are  to  be  added 

i  are  prepared  in  the  same  manner  as  nutrient  broth,  adding  i 
per  cent,  of  dextrose,  lactose,  saccharose  or  other  sugar.  The 
sugar  is  to  be  added  before  sterilizing.  Sterilizing  in  the  auto- 
clave is  to  be  preferred  because  the  longer  steam  sterilization  is 
apt  to  cause  inversion  of  the  sugar.  The  reaction  of  the  sugar 
broths  shall  be  neutral  to  phenolphthalein. 

These  media  are  much  used  in  testing  for  the  presence  of 
Bacillus  coli  (dextrose  broth).  The  committee  states  that  the 
removal  of  muscle  sugar  by  inoculating  with  B.  coli  is  not  nec- 
essary if  small  amounts  of  gas  formation  are  to  be  disregarded. 
In  the  routine  work  of  testing  water  for  the  presence  of  the  B. 
coli  a  sufficient  volume  of  the  water  to  be  tested  is  added  so  that 
the  resulting  mixture  will  be  one  of  normal  strength.  The  com- 
mittee also  advises  against  the  use  of  beef  extracts  in  place  of  the 
laboratory  made  beef  infusions. 

3.  Nutrient  Gelatin. — Make  the  beef  infusion  in  the  manner  al- 
ready described.  After  the  first  filtering  through  cotton  or  cotton 
flannel,  add  10  per  cent,  of  gelatin  (the  per  cent,  being  based  on  the 
weight  of  the  beef  infusion  instead  of  volume  and  the  weight  of 
the  gelatin  to  be  on  a  basis  of  dry  condition,  and  i  per  cent,  of 
peptone)  and  warm  over  water  bath  with  constant  stirring  until 
the  peptone  and  gelatin  are  entirely  dissolved.  While  dissolving 
the  peptone  and  gelatin  the  temperature  should  not  rise  above 
60°  C.  Boil  for  2  min.  and  adjust  the  reaction  to  -f-i.oo  per 
cent.  Heat  for  40  min.  over  water  bath  or  in  steam  sterilizer 
and  restore  any  loss  by  evaporation.  Again  adjust  the  reaction 
if  necessary  and  boil  over  open  flame  for  5  min.  with  constant 
stirring.  Restore  loss,  filter  until  clear,  titrate  and  record  this 
final  reaction.  Tube  and  sterilize  as  for  beef  broth  and  at  once 
store  in  ice  chest.  Protect  against  evaporation  as  already 
explained. 

4.  Nutrient  Agar. — Boil  15  grams  (dry  weight)  of  washed 
thread  agar  in  500  cc.  of  distilled  water  for  half  an  hour  and  make 


78  BACTERIOLOGICAL   METHODS 


^ 


up  weight  to  500  grams.  Infuse  500  grams  of  lean  meat  in  500 
cc.  of  distilled  water  for  24  hr.  in  ice  chest.  Make  up  loss  by 
evaporation,  strain,  weigh  filtered  infusion  and  add  2  per  cent, 
of  peptone.  Warm  on  water  bath  with  constant  stirring  until  all 
of  the  peptone  is  dissolved.  To  500  grams  of  the  meat  infusion  add 
500  cc.  of  the  3  per  cent,  agar  solution,  keeping  the  temperature 
below  60°  C.  Boil  for  i  min.  and  titrate  to  +i-o.  Sterilize 
in  steam  for  40  min.  and  restore  any  loss  by  evaporation.  Re- 
adjust if  necessary  and  then  boil  for  5  min.  with  constant  stirring. 
Restore  any  loss  due  to  evaporation  and  filter  by  passing  it  through 
the  filtering  material  (cotton  and  cotton  flannel  or  perforated 
filter  paper)  at  least  three  times.  Titrate  and  record  the  final 
reaction.  Tube,  sterilize  and  store  as  for  gelatin  media.  It 
must  be  borne  in  mind  that  agar  media  are  never  as  clear  as  broth 
or  gelatin  media. 

5.  Lactose  Litmus  Agar. — To  make  this  medium  add  i  per  cent, 
of  lactose  to  nutrient  agar  just  before  sterilizing  and  make  the 
reaction  neutral  to  phenolphthalein.  ■ 

If  this  medium  is  to  be  used  in  tubes  the  sterilized  azolitmin 
(i  per  cent,  aqueous  solution)  is  added  just  before  the  final  mass 
sterilization,  that  is,  the  sterilization  before  pouring  into  the 
tubes. 

If  the  medium  is  to  be  used  in  Petri  dishes,  the  azolitmin 
not  added  until  ready  to  pour  into  the  dishes. 

The  azolitmin  and  the  lactose  should  be  sterilized  separately 
before  adding  to  the  agar  medium,  though  it  is  permissible  to  mix 
the  lactose  with  the  agar  and  sterilize  together,  preferably  in  the 
autoclave  (120°  C.  for  15  min.). 

It  would  appear  that  the  azolitmin  of  the  market  varies  con- 
siderably and  many  bacteriologists  prefer  the  pure  litmus.     A  i 
per  cent,  aqueous  suspension  of  azolitmin  should  dissolve  readil 
when  boiled  for  5  min. 

This  medium  is  much  used  in  bacteriological  work  on  pr 
sumptive  sewage  contaminations,  as  estimating  the  temperatur 


le 

I 


1 


STANDARD    MEDIA  79 

differential  colonies  (20°  C.  and  37°  C),  red  colonies  and  total 
colonies,  etc. 

6.  Lactose  Bile. — This  medium  is  to  be  made  in  two  ways: 
Add  I  per  cent,  of  peptone  and  i  per  cent,  of  lactose  to  sterilized 
undiluted  fresh  ox  gall;  or  add  the  peptone  and  lactose  to  a  10 
per  cent,  aqueous  solution  of  freshly  made  dry  ox  gall.  It  is  used 
without  titrating.  Old  dried  ox  gall  should  not  be  used.  Obtain 
it  from  a  reliable  dealer.  If  possible,  make  arrangements  to  get 
the  fresh  undiluted  ox  gall  from  some  abattoir. 

This  is  the  standard  medium  for  making  the  quantitative  as 
well  as  qualitative  tests  for  the  colon  group  of  bacilli. 

7.  Liver  Broth. — Chop  500  grams  of  fresh  beef  liver  into  small 
pieces  and  place  in  1000  cc.  of  distilled  water.  Weigh  infusion 
and  container.  Boil  for  2  hr.  in  rice  cooker,  starting  cold 
and  stirring  occasionally.  Make  up  loss  in  weight  and  pass 
through  wire  or  cloth  strainer.  Add  10  grams  of  peptone,  10 
grams  of  dextrose  and  i  gram  of  di-potassium  phosphate  (K2- 
HPO4) .  Dissolve  the  added  ingredients  by  warming  in  rice  cooker 
with  stirring  and  then  titrate  to  the  neutral  point  (to  phenol- 
phthalein).  Boil  for  30  min.  in  the  rice  cooker  and  for  5  min. 
over  open  flame  with  constant  stirring  to  prevent  the  carameliza- 
tion  of  the  dextrose.  Make  up  loss  due  to  evaporation  and  filter. 
Tube,  sterilize  and  store  as  for  other  media. 

This  is  a  much  used  enriching  medium  which  gives  asg  forma- 
tion with  all  of  the  species  which  ferment  dextrose.  It  is  also 
much  used  to  rejuvenate  pure  cultures  of  bacteria  and  encourages 
the  development  of  attenuated  forms  of  bacteria. 

8.  Hiss  Typhoid  Bacillus  Medium  .—Two  media  are  used.  One 
for  the  isolation  of  the  typhoid  bacillus  by  the  plating  method,  and 
the  other  for  the  differentiation  of  the  typhoid  germ  from  other 
forms  in  tube  cultures.  The  former  is  designated  as  the  plate 
medium  and  the  second  as  the  tube  medium.  .They  are  prepared 
as  follows: 


8o  BACTERIOLOGICAL  METHODS 

a.  Plate  Medium 

Agar lo  grams 

Gelatin 25  grams 

Salt 5  grams 

Liebig's  meat  extract 10  grams 

Dextrose 10  grams 

Water  (distilled) 1000  cc. 

Add  gelatin  when  the  agar  is  melted,  dissolve  the  gelatin,  add 
the  other  ingredients,  titrate  to  +2.0  per  cent.,  filter,  etc.,  as  for 
other  media.  The  medium  is  to  be  clarified  by  adding  the  whites 
of  one  or  two  eggs,  well  beaten  in  25  cc.  of  distilled  water,  boil  for 
45  min.  and  filter  through  absorbent  cotton.  Do  not  add  the 
dextrose  until  after  clearing. 

b.  Tube  Medium 

Agar 5  grams 

Gelatin 80  grams 

Salt 5  grams 

Liebig's  meat  extract 5  grams 

Dextrose 10  grams 

Water  (distilled") 1000  ec. 

The  manner  of  preparation  is  the  same  as  for  the  plate  medium. 
However,  the  reaction  is  to  be  +1.5  instead  of  +2.0  per  cent. 
Without  the  dextrose  and  less  salt  and  titrated  to  +1.0  per 
cent.,  the  plate  medium  constitutes  the  ordinary  nutrient  agar- 
gelatin  medium  which  was  formerly  very  much  used  because  it 
possessed  the  solidifying  properties  of  agar  combined  with  the 
nourishing  properties  of  gelatin. 

9.  Endo  Medium. — This  medium  is  much  used  in  testing  for 
the  colon  bacillus.  It  is  variously  modified  by  different  workers 
and  it  is  highly  important  that  some  standard  method  of  pre- 
paring the  medium  should  be  adopted  and  adhered  to.  The 
following  is  the  method  of  preparation  and  use  recommended  by 
the  committee. 

Add  30  grams  of  powdered  agar  to  i  liter  of  cold  water  by 
sifting  slowly  upon  the  surface  of  the  water  and  allowing  it  to 


STANDARD   MEDIA  8 1 

settle.  Add  lo  grams  of  peptone  and  5  grams  of  Liebig^s  meat 
extract.  Heat  in  rice  cooker  until  the  ingredients  are  entirely 
dissolved.  Neutralize  with  sodium  carbonate,  using  litmus  as 
an  indicator,  and  then  add  10  cc.  of  a  10  per  cent,  solution  of 
sodium  carbonate. 

Store  the  medium  in  lots  of  100  cc.  using  flasks  large  enough 
to  permit  the  addition  of  the  other  ingredients.  Sterilize  for  2 
hr.  in  streaming  steam. 

To  use  the  Endo  medium  proceed  as  follows:  Make  a  10 
per  cent,  aqueous  solution  of  sodium  sulphite  and  add  2  cc.  of 
fuchsin  solution  (10  per  cent,  of  basic  fuchsin  in  96  per  cent, 
alcohol)  to  10  cc.  of  the  sulphite  solution  and  steam  this  mixture 
for  a  few  minutes  in  the  steam  sterilizer.  Add  i  gram  of  chemically 
pure  sterilized  lactose  to  each  100  cc.  of  the  Endo  medium  after 
the  medium  has  been  liquefied  and  while  the  temperature  is  not 
above  60°  C.  While  the  medium  is  still  liquid,  add  0.5  cc.  of 
the  fuchsin-sulphite  solution  and  then  pour  into  the  Petri  plates 
and  allow  to  harden  in  the  incubator.  The  sulphite  solution 
must  be  prepared  fresh  as  needed. 

10.  Milk. — The  milk  to  be  used  for  cultural  purposes  must  be 
pure  and  recently  drawn.  In  all  cases  the  milk  of  the  grade  or 
quality  known  as  "certified  milk"  is  to  be  preferred.  The 
recently  drawn  milk  is  to  be  placed  in  the  refrigerator  for  12 
hr.,  so  as  to  permit  the  cream  to  rise  to  the  top  and  any  sus- 
pended matter  to  sink  to  the  bottom.  Skim  the  milk  and  siphon 
off  all  but  the  bottom  sedimentary  portion.  Adjust  to  +1.0  per 
cent.     Tube  and  sterilize. 

Litmus  milk  is  made  by  adding  i  per  cent,  of  sterilized  azo- 
litmin  to  the  above.  In  using  litmus  milk  always  set  aside  a 
control  tube  with  the  inoculated  tubes  for  purposes  of  color 
comparison. 

Because  of  the  difficulty  of  always  getting  a  uniformly  high 
quality  of  cow^s  milk,  it  has  been  suggested  that  an  artificial 
substitute    be    employed.     Hill    and    his    pupils    recommend    a 


82  BACTERIOLOGICAL   METHODS 

medium  in  which  prepared  casein  (nutrose)  is  the  principle  in- 
gredient. Chemically,  nutrose  is  a  caseinate  of  sodium  and  is 
prepared  as  follows:  Moist  casein  precipitated  from  skimmed 
milk  is  washed  with  water  in  a  solution  of  sodium  hydroxide, 
evaporating  the  solution  to  dryness  in  vacuo,  powdering  the  residue 
and  washing  successively  with  alcohol  and  ether  and  then  dry- 
ing. It  is  a  coarse,  white,  odorless  and  tasteless  powder,  forming 
a  turbid  adhesive  solution  with  water,  having  an  alkaline  reac- 
tion toward  litmus  and  an  acid  reaction  toward  phenolphthalein. 
It  is  a  food  product  intended  for  the  sick  because  of  its  easy  di- 
gestion. It  is  made  in  Germany  but  may  be  secured  through  any 
of  the  larger  American  pharmaceutical  houses  (Victor  Koechl  & 
Co.,  New  York  City). 

The  formula  for  making  the  artificial  milk  is  as  follows: 

Nutrose 24  grams 

Lactose 10  grams 

Distilled  water 1000  cc. 

Dissolve  the  nutrose  and  lactose  in  the  water  (cold)  for 
12  hr.  with  occasional  thorough  shaking  and  then  filter 
through  cotton.  Tube  and  sterilize  at  110°  C.  for  20  min.,  or 
in  the  steam  sterilizer  in  the  usual  manner.  No  adjustment  is 
required. 

This  medium  contains  all  of  the  nutritive  ingredients  of  cow's 
milk  with  the  exception  of  fat  which  is  not  desired  for  the  ordinary 
cultural  work.  It  is  of  uniform  quality  and  is  said  to  give  far 
more  uniform  results  than  cow's  milk.  It  is  furthermore  more 
translucent  than  cow's  milk  and  shows  the  reactions  with  in- 
dicators much  better.  It  would  be  advisable  to  make  the  artificial 
milk  the  standard  substitute  for  cow's  milk. 

II.  Peptone  Medium. — This  is  simply  a  i  per  cent,  peptone 
solution  in  distilled  water  and  is  intended  to  be  used  for  making 
the  indol  test.  Beef  broth  from  which  muscle  sugar  has  been 
removed  by  inoculating  with  B.  coli  is  believed  to  be  objection- 


STANDARD   MEDIA  S^ 

able  because  of  the  toxins  present  and  which  interfere  with  the 
growth  of  many  species  of  bacteria. 

Other  media  of  a  more  or  less  special  character  will  be  described 
or  referred  to  under  the  discussion  of  methods.  Those  described 
above  are  the  more  important  ones  required  in  the  bacteriological 
examination  of  foods  and  drugs. 

7.  Technique  for  Making  Quantitative  and  Qualitative  Estimations 
by  the  Plating  Methods 

As  has  been  explained,  the  plating  method  is  intended  to  de- 
termine the  number  of  living  bacteria  present  in  foods  and  drugs 
and  the  results  supplement  the  results  of  the  method  of  making 
the  direct  counts  already  described.  From  this  statement  it  is 
evident  that  the  quantitative  results  by  the  two  methods  are  not 
the  same.  For  example,  the  bacterial  count  of  a  catsup  by  the 
direct  method  may  be  very  high  while  the  plating  method  may 
give  negative  results,  due  to  the  fact  that  the  heat  sterilization 
employed  at  the  cannery  killed  all  of  the  bacteria  present.  This 
also  shows  why  it  is  absolutely  necessary  to  employ  both  methods 
in  order  to  form  a  correct  estimate  of  the  total  contamination  of 
the  substance. 

The  following  suggestions  on  laboratory  technique  are  given 
with  a  view  to  the  unification  of  methods,  thereby  leading  to 
greater  uniformity  in  comparative  results. 

I.  Apparatus. — Test-tubes  to  be  used  for  the  usual  cultural 
purposes  shall  be  of  medium  weight  and  thickness,  15  cm.  long 
by  1.6  cm.  diam.  Petri  dishes  shall  be  10  cm.  in  diam.  Petri 
dishes  with  porous  covers  are  preferred.  All  glass  ware  must 
be  scrupulously  clean  and  may  be  sterilized  by  exposing  to  a  dry 
heat  of  about  150°  C.  for  a  period  of  i  hr.,  after  being  cleaned, 
wiped  dry  and  plugged  with  a  good  grade  of  commercial  cotton. 
A  browning  of  the  free  ends  of  the  cotton  plugs  indicates  that 
the  right  degree  of  heating  has  been  attained.  A  standard  wire 
7 


84  BACTERIOLOGICAL   METHODS 

loop  is  made  as  follows:  Bend  the  end  of  a  No.  27  platinum  wire, 
10  cm.  long,  around  a  piece  of  No.  10  wire.  The  free  portion  of 
the  straight  platinum  wire  inoculating  needle,  shall  be  10  cm. 
long  (No.  27  wire).  The  standard  fermentation  tube  shall  be 
of  the  following  proportions.  The  length  of  the  closed  end  of  the 
fermentation  tube  (diameter  about  1.5  cm.)  shall  be  about  14  cm., 
and  the  open  end  shall  be  of  bulbous  form  (diameter  of  bulb  about 
3.8  cm.)  large  enough  to  hold  all  of  the  liquid  in  the  closed  end. 
Larger  and  smaller  fermentation  tubes  than  the  standard  just 
described  may  be  used  for  special  purposes.  Standard  and  other 
fermentation  tubes  may  or  may  not  be  graduated  as  the  special 
purposes  may  require. 

2.  Amounts  of  Media  to  be  Tubed. — The  standard  amount  of 
culture  medium  to  be  placed  in  each  test-tube  of  standard  size  is 
5  and  10  cc,  the  media  to  be  introduced  by  means  of  a  suitable 
burette.  Greater  or  lesser  quantities  may  be  used  as  occasion 
may  require.  Tubes  containing  just  10  cc.  of  culture  media  are 
required  for  the  plating  purposes.  5  cc.  quantities  (of  gelatin, 
agar  and  other  solid  media)  are  required  for  making  slants. 

3.  Amounts  of  Culture  Media  to  be  Plated. — For  the  usual 
quantitative  determinations  by  the  plating  method,  10  cc.  of  the 
culture  medium  shall  be  poured  in  each  standard  Petri  dish. 

The  required  number  of  tubes  each  containing  10  cc.  of  agar  or 
gelatin  culture  medium  are  placed  in  the  steam  sterilizer  until 
the  medium  is  entirely  liquefied  and  then  placed  in  a  beaker  or  other 
suitable  container  with  lukewarm  water,  with  thermometer. 
Plate  the  gelatin  medium  when  the  thermoineter  registers  be- 
tween 25°  and  30°  C.  The  temperature  of  the  medium  must  not 
be  more  than  30°  C.  If  the  temperature  is  less  than  25°  C.  the 
gelatin  will  begin  to  coagulate  and  will  not  pour  and  spread 
properly.  Agar  media  must  be  plated  at  a  higher  temperature 
than  gelatin  media,  usually  40°  to  42°  C.  The  Petri  dishes  should 
be  warm  when  the  media  are  poured,  the  temperature  being  ap- 
proximately the  same  as  that  of  the  medium  when  it  is  poured. 


TECHNIQUE  85 

This  will  insure  a  more  uniform  spreading  of  the  medium  over  the 
bottom  of  the  dish. 

To  pour  the  liquefied  agar  or  gelatin  from  the  tubes,  remove  the 
cotton  plug  and  flame  the  mouth  of  the  tube  so  as  to  kill  any 
bacteria  or  spores  that  may  be  present;  raise  one  side  of  the  cover 
just  high  enough  to  permit  bringing  the  tube  to  the  middle  of  the 
dish  and  pour  contents  into  the  dish  over  the  material  planted  into 
the  middle  of  the  dish.  Let  cover  of  the  dish  sink  into  place  and 
by  very  slight  tilting  of  the  Petri  dish  induce  the  culture  medium 
to  spread  evenly  over  the  bottom  of  the  dish  before  the  medium 
has  had  time  to  coagulate.  As  the  medium  spreads  it  also  causes 
the  spreading  of  the  planted  material. 

Many  workers  use  5  cc.  of  the  medium  for  plating,  instead  of 
10  cc.  as  above  recommended.  The  smaller  amount  is  satisfactory 
when  I  cc.  quantities  are  to  be  planted  or  inoculated.  However, 
in  order  to  make  sure  that  the  entire  area  of  the  bottom  of  the 
dish  is  well  covered,  10  cc.  quantities  should  be  used.  The  larger 
amount  also  minimizes  the  influences  which  the  changes  in 
evaporation  in  the  media  may  have  upon  the  quantitative  results. 

4.  Method  of  Making  the  Plate  Cultures. — Absolutely  clean 
sterilized  (dry  heat  of  150°  C.  for  i  hr.)  Petri  dishes  of  the 
standard  size  (10  cm.  diam.)  are  used,  o.i  cc.  quantities  of 
the  substance  to  be  cultured,  or  dilutions  thereof,  are  planted  or 
delivered  into  the  middle  of  the  dish,  an  absolutely  clean  and 
sterile  i  cc,  pipette  accurately  divided  into  tenths.  The  cover  of 
the  dish  is  to  be  lifted  just  high  enough  to  permit  placing  the  pipette 
in  position,  and  is  to  be  replaced  just  as  soon  as  possible. 

In  the  usual  water  analysis  work,  i  cc.  quantities  are  generally 
planted,  instead  of  o.i  cc.  quantities  as  above  recommended. 
For  purely  quantitative  results,  the  smaller  amounts  should  be 
planted  because  the  larger  amounts  may  include  enough  of  the 
inoculating  liquid  to  interfere  with  the  uniformity  of  results. 

Formerly  it  was  customary  to  mix  the  material  to  be  planted 
with  the  medium  in  the  tube  before  plating.     This  method  has 


86  BACTERIOLOGICAL  METHODS 

some  very  objectionable  features,  chief  of  which  is  that  the  residue 
remaining  in  the  tube  after  pouring  retained  a  certain  percentage 
of  the  organisms,  thus  interfering  with  the  accuracy  of  the  results. 
It  must,  however,  be  admitted  that  the  method  has  some  ad- 
vantages, chief  of  which  is  the  more  uniform  mixing  of  the  bacteria 
with  the  medium  and  their  more  uniform  distribution  in  the 
plate,  making  accurate  counting  of  the  colonies  easier. 

5.  Making  the  Dilutions. — Whether  or  not  making  dilutions 
is  necessary  depends  upon  the  number  of  organisms  present  in 
the  substance  to  be  analyzed.  The  number  of  colonies  in  a 
Petri  dish  must  not  exceed  200  in  order  to  make  counting  fairly 
easy  and  accurate.  In  fact  with  the  method  of  direct  planting, 
as  usually  recommended,  which  generally  results  in  a  some- 
what irregular  distribution  of  the  bacteria  (hence  also  the  colonies 
to  be  counted)  it  would  be  desirable  to  make  the  dilutions  such 
that  the  number  of  colonies  in  each  plate  shall  not  exceed  100. 
If  0.1  cc.  quantites  are  to  be  plated  or  planted,  as  above  recom- 
mended, it  would  follow  that  dilution  would  not  be  necessary 
as  long  as  'the  number  of  bacteria  per  cc.  does  not  exceed 
1000. 

However,  since  most  food  and  drugs  contain  more  chan  that 
number  of  bacteria  per  cc,  it  becomes  necessary  to  make  dilu- 
tions. The  standard  dilutions  are  made  by  tens,  as  i-io,  i-ioo, 
i-iooo,  and  1-10,000.  The  dilutions  are  made  by  adding  i  cc. 
of  the  substance  to  be  analyzed  to  9,  99,  999  and  9999  cc.  of 
sterile  distilled  water,  or  other  desirable  sterile  diluent,  and 
shaking  thoroughly.  In  practice  it  is  desirable  to  plate  three 
of  the  graded  dilutions,  so  that  the  second  higher  dilution  will  in 
all  probability  yield  about  100  bacteria  in  the  o.i  cc.  of  the 
material  plated.  Thus  with  fairly  pure  drinking  water,  the  plant- 
ings would  be  made  from  the  undiluted  water,  the  i-io  and  the 
i-ioo  dilutions,  presuming  that  there  are  about  10,000  bacteria 
per  cc.  present.  In  case  of  unusually  pure  drinking  water,  that  is 
water  in  which  the  number  of  bacteria  is  probably  not  more 


TECHNIQUE 


87 


than  50  per  cc,  it  would  be  desirable  to  use  i  cc.  quantities  for 
plating  which  would  give  about  50  colonies  in  the  plate. 

The  thorough  mixing  of  the  sample  before  making  the  dilutions 
is  of  the  greatest  importance,  likewise  the  thorough  rriixing  of 
each  dilution  before  taking  out  the  quantity  to  be'plated.     Each 


g 


y. 


W 


Fig.  23. — Types  of  growth  in  stab  cultures.  A,  Non-liquefying,  i,  Filiform 
{Bacillus  coli);  2,  beaded  (Streptococcus  pyogenes);  3,  echinate  (Bacterium  acidi 
lactici);  4,  villous  (Bacterium  murisepticum);  5,  arborescent  (Bacillus  mycoides). 

By  Gelatin  liquefying.  6,  Crateriform  (Bacillus  vulgare,  24  hr.;  7,  napiform 
(Bacillus  subtiliSf  48  hr.);  8,  infundibuliform  (Bacillus  prodigiosus);  9,  saccate 
(Micros pir a  finkleri);  10,  stratiform  (Pseudomonas  fiuorescens). — (McFarland, 
after  Frost.) 

test  should  be  made  in  triplicate,  taking  up  the  i  cc.  amounts 
for  making  the  dilutions  with  three  different  clean  sterile  pipettes. 
It  is  preferable  to  use  a  new  pipette  for  each  dilution.  If  it  is 
not  convenient  to  have  on  hand  a  sufficient  number  of  clean 
sterilized    pipettes,    the    pipette    in    use    must   be   thoroughly 


88  BACTERIOLOGICAL   METHODS 


y  of  disH 


rinsed  in  sterilized  distilled  water,  using  a  fresh  supply 
tilled  water  for  each  rinsing. 

Draw  the  liquid  to  be  plated  into  the  pipette,  place  thumb 
over  the  upper  end  of  pipette,  let  liquid  run  out  until  one  of  the 
O.I  cc.  marks  is  reached,  then  bring  the  lower  end  of  the  pipette 
close  to  the  surface  of  the  liquid  in  the  diluting  tube  and  let 
just  O.I  cc.  run  out,  the  finer  degrees  of  accuracy  are  attained 
by  using  or  not  using  the  meniscus  of  the  liquid  projecting  from 
the  lower  end  of  the  pipette  after  the  last  drop  has  fallen.  This 
correcting  droplet  is  secured  by  touching  the  lower  end  of  the 
pipette  lightly  against  the  inside  of  the  diluting  tube  at  a  point 
near  the  surface  of  the  liquid,  without,  however,  actually  touch- 
ing the  liquid.  A  similar  adjustment  may  be  made  when  taking 
up  the  I  cc.  amount  to  be  diluted,  only  in  this  case  the  pipette  is 
of  course  to  be  touched  against  the  side  of  the  tube  or  vessel 
containing   the   liquid   of   which    the   dilution   is    to  be  made. 

Thorough  mixing  of  the  contents  of  the  diluting  tube  is  at- 
tained by  vigorous  shaking.  Place  the  thumb  over  the  open- 
ing of  the  tube,  interposing  a  piece  of  sterilized  rubber  sheeting 
such  as  is  used  by  dentists.  Some  workers  mix  the  contents  of 
the  tube  by  rapidly  rotating  between  the  two  hands  and  by  tap- 
ping against  the  palm  of  one  hand. 

6.  Incubation. — The  regulation  incubators  are  to  be  used. 
It  is  highly  important  that  there  should  be  ample  ventilation,  a 
matter  to  which  amateurs  and  even  experienced  bacteriologists 
as  a  rule  give  little  or  no  attention.  All  modern  incubators  are 
supplied  with  ventilating  openings  at  the  top  which  should  be 
kept  open  most  of  the  time.  The  air  in  the  incubating  chamber 
should  be  practically  saturated  with  moisture,  which  may  be 
accomplished  by  placing  a  flat  dish  containing  water  in  the  lower 
chamber. 

Two  standard  incubating  temperatures  are  employed,  namely, 
20°  C.  and  37°  C,  the  first  corresponding  to  the  ordinary  room 
temperature  and  the  second  to  the  body  (human)  temperature 


TECHNIQUE 


89 


or  blood  heat.  The  devices  to  regulate  the  temperature  should 
be  such  that  the  variation  from  the  two  standards  given  shall  not 
be  more  than  2°,  that  is,  not  more  than  1°  in  either  direction. 

There  is  no  standard  time  of  incubation.  For  work  in  the 
study  of  water  sanitation  as  carried  out  in  Germany,  England 
and  also  in  the  United  States,  gelatin  plates  are  incubated  for  2 
days  at  a  temperature  of  20°  C.  It  is  suggested  that  the  period 
be  extended  to  3  days   in  order  to  get  more  accurate  results. 


',  "^^^ 


V.     \ 


I 


Fig.  24. — Types  of  streak  culture,  i,  Filiform  (B.  coli);  2,  echinulate  {B.  acidi 
laciici);  3,  beaded  (Str.  pyogenes);  4,  effuse  (B.  vulgaris)',  5,  arborescent  (Bacillus 
mycoides). — {McFarland  after  Frost.) 

From  I  to  2  days  is  the  usual  time  of  exposure  for  the  higher 
temperature  (37°  C). 


8.  Practical  Application  of   the  Quantitative  Estimations   by  the 

Plating  Methods 

The  relative  importance  of  the  quantitative  bacteriological  de- 
terminations by  the  method  of  direct  counting  and  by  the  plating 
method  has  been  explained.  Both  methods  must  be  made 
standard  in  every  food  and  drug  laboratory.  Quantitative  esti- 
mations by  the  plating  method  should  take  precedence  with  all 
substances  containing  largely  living  organisms  such  as  water 
supplies  of  all  kinds,  milk,  raw  meats,  and  shellfish,  etc.,  and  all 
substances  in  which  infection  is  suspected,  even  though  such  sub- 


go  BACTERIOLOGICAL  METHODS 


stances  may  have  been  subjected  to  processes  of  sterilization 
some  phase  of  the  processing  or  of  manufacture. 

In  a  general  way  the  quantitative  results  by  the  plating  metho(MJ 
are  to  be  interpreted  in  a  manner  similar  to  the  results  by  the 
direct  count.  In  some  cases  the  question  at  issue  may  be  relative 
to  the  presence  or  absence  of  viable  bacteria  in  substances  which 
presumably  do  not  contain  living  organisms,  such  as  canned  foods 
generally.  Manufacturers  of  canned  products  are  of  the  opinion 
that  the  methods  of  heat  sterilization  employed  will  kill  all  of  the 
bacteria  which  may  have  been  present  at  the  time  of  canning. 
This  is  undoubtedly  true  in  many  cases,  but  in  other  instances  it  is 
only  too  evident  that  retarded  fermentation  processes  continue 
after  the  cans  are  sealed,  which  accounts  for  the  high  counts  in 
canned  food  products  which  contained  only  small  numbers  of  bac- 
teria at  the  time  the  cans  were  sealed.  These  subdued  fermenta- 
tion processes  as  a  rule  do  not  result  in  the  formation  of  sufficient 
gas  to  produce  "swells"  anii  hence  the  article  is  not  suspected 
until  the  container  is  opened  when  a  more  or  less  disagreeable  or 
peculiar  odor  is  noticeable,  which  is,  as*a  rule,  not  sufficiently 
pronounced  to  prevent  the  use  of  the  article  as  food. 

In  addition  to  the  purely  quantitative  results,  the  plating 
method  indicates  the  general  qualitative  character  of  .the  organ- 
isms present  and  conveys  some  idea  as  to  the  course  of  the  infection 
or  contamination  as  shown  by  the  characters  of  the  colonies  de- 
veloped in  the  Petri  dish  or  in  the  tubes. 

9.  Qualitative  Determinations 

The  chief  qualitative  determinations  in  food  and  drugs  labora- 
tories pertain  to  sewage  contamination.  The  recognition  and 
determination  of  pathogenic  bacteria  as  the  typhoid  bacillus, 
the  cholera  bacillus,  diphtheria  bacillus,  etc.,  is  an  incidental 
possibility  in  the  food  laboratory  routine  and  not  a  regular  part 
of  it.     Of  far  greater  significance  is  the  recognition  of  the  evidence 


QUALITATIVE   DETERMINATIONS  9I 

of  the  presence  of  intestinal  parasites,  such  as  the  segments  of  tape 
worm,  the  larvae  and  ova  of  such  parasites,  etc.,  as  already  stated 
under  the  discussion  of  the  direct  method  of  making  counts.  The 
bacteriologists  in  food  and  drugs  laboratories  should  be  qualified 
to  recognize  all  of  the  possible  disease  germs  and  the  smaller  carriers 
of  disease  which  may  be  associated  with  food  substances  and  they 
should  be  able  to  demonstrate  the  presence  of  such  contamination, 
if  necessary.  The  thus  far  recognized  routine  in  food  and  drugs 
laboratories  and  in  public  health  laboratories  is  limited  to  making 
the  so-called  presumptive  colon  bacillus  test,  as  indicative  of  sewage 
contamination  or  contamination  with  fecal  matter.  Sewage  con- 
tamination means  primary  contamination  with  fecal  matter.  The 
reason  why  the  colon  bacillus  test  has  been  selected  as  giving  satis- 
factory evidence  of  sewage  contamination  is  because  this  bacillus 
is  most  abundant  and  is  constantly  present  in  fecal  matter.  Any 
considerable  number  of  colon  bacilli  in  water  supplies  or  in  food 
substances  is  evidence  of  gross  negligence  and  defects  as  to  sanitary 
requirements. 

As  far  as  the  practical  work  in  finding  evidence  of  the  sewage 
contamination  of  food  substances  is  concerned,  there  is  no  effort 
made  to  isolate  and  identify  a  definite  bacillus  recognizable  as 
Bacillus  coll.  It  is  rather  the  recognition  of  certain  cultural 
characteristics  which  have  come  to  be  recognized  as  being  peculiar 
to  the  bacilli,  known  as  the  B.  colt  group,  all  of  which  are  traceable 
to  intestinal  origin.  Furthermore  this  group  of  bacteria  is  very 
widely  distributed  in  the  animal  kingdom,  being  in  no  wise  limited 
to  the  intestinal  tract  of  man.  The  B.  coli  group  of  the  lower 
animals  is  in  all  probability  different  from  that  which  inhabits 
man  and  certain  workers  have  made  attempts  to  differentiate 
them  by  means  of  special  cultural  methods,  but  thus  far  these 
methods  are  not  sufficiently  perfected  to  be  used  practically  in 
food  and  drugs  laboratories.  These  statements  also  apply  to  the 
Streptococci  group  of  intestinal  origin.  However,  some  of  the 
laboratory  results  thus  far  attained  would  indicate  that  in  the 


92  BACTERIOLOGICAL  METHODS 

near  future  it  will  be  possible  to  differentiate  between  pollutions 
traceable  to  human  origin  and  such  as  are  traceable  to  cow,  horse 
or  hog  manure,  for  example.  It  cannot  be  denied  that  food 
materials  intended  for  human  consumption  which  are  contamin- 
ated to  any  distinctly  appreciable  amount  with  the  contents  of 
the  intestinal  tract  of  any  animal,  are  unsanitary  and  hence  unfit 
for  human  consumption. 

In-as-much  as  the  intestinal  bacteria  (bacilli  and  streptococci) 
are  very  abundant  and  very  widely  distributed,  it  is  quite  evident 
that  it  would  be  impracticable  to  pronounce  all  foods  unfit  for 
use  if  only  one  or  a  few  intestinal  organisms  were  found  to  be  present 
in  a  comparatively  large  quantity.  Human  feces  contains  about 
one-third  bacteria  (dry  weight),  the  majority  of  which  belong  to  the 
colon  group,  and  the  exterior  of  the  human  body  carries  bacteria 
derived  from  the  intestinal  tract,  especially  the  hands  and  the 
deposits  under  the  finger  nails.  Flies  are  carriers  and  distributors, 
of  intestinal  bacteria.  The  dust  of  the  streets  and  street  sweepings 
contain  large  numbers  of  bacteria  derived  from  the  intestinal  tract 
of  the  horse,  etc.  It  would  be  impracticable  to  enter  into  a 
fuller  discussion  of  the  distribution  of  bacteria  traceable  to  in- 
testinal origin.  Suffice  it  to  state  that  it  is  the  work  of  the  food 
bacteriologist  to  determine  the  presence,  in  articles  intended  as 
food,  of  those  bacteria  which  indicate  contamination  with  fecal 
matter,  no  matter  what  the  source  of  such  objectionable  matter 
may  be.  The  basis  for  the  condemnation  of  contaminated  foods  is 
quantitative  in  the  comparative  sense.  For  example,  the  finding 
of  a  few  colon  bacilli  in  large  quantities  of  water  or  their  occasional 
presence  in  small  quantities  of  water,  does  not  indicate  that  the 
water  is  unsuitable  for  drinking  purposes.  If,  however,  the  colon 
bacilli  appear  in  a  large  proportion  of  many  small  samples  (i  cc. 
or  less)  of  water  it  is  safe  to  conclude  that  there  is  considerable 
recent  sewage  contamination  and  that  such  water  is  dangerous 
to  health.  The  intestinal  bacteria  are  in  themselves  not  seriously 
pathogenic  to  man  even  when  taken  in  considerable  numbers. 


QUALITATIVE   DETERMINATIONS  93 

The  real  source  of  danger  lies  in  the  fact  that  the  intestinal  bacteria 
normal  to  man  and  the  lower  animals,  may  be  and  frequently  are 
associated  with  pathogenic  bacteria,  such  as  the  typhoid  bacillus 
and  the  dysentery  bacillus.  Our  long  experience  with  the  con- 
sumption of  sewage  contaminated  water  supplies,  has  shown  that, 
as  a  rule,  the  first  danger  sign  of  the  excessive  contamination  was 
usually  a  marked  increase  in  the  number  of  cases  of  dysentery, 
generally  followed  by  sporadic  cases  and  epidemics  of  typhoid 
fever. 

The  practical  results  of  the  quantitative  bacterial  determi- 
nations of  food  substances  combined  with  the  qualitative  tests 
for  the  colon  group,  has  proven  of  the  highest  value  and  it  is 
considered  entirely  feasible  to  continue  the  application  of  the 
tests  and  to  suggest  ways  and  means  of  improving  the  laboratory 
technique  covering  such  methods.  The  qualitative  methods 
thus  far  worked  out  are  based  upon  a  knowledge  of  the  life  history 
of  the  bacteria  concerned  and  may  be  briefly  stated  as  follows: 
Normal  intestinal  bacteria  and  such  other  bacteria  as  may  develop 
in  the  intestinal  tract,  such  as  the  typhoid  bacillus,  the  cholera 
bacillus,  the  dysentery  bacilli,  etc.,  are  adapted  to  a  temperature  of 
about  37°  C.  and  they  feed  upon  the  food  materials  found  in  the 
intestinal  tract  and  have  a  somewhat  reduced  oxygen  supply. 
Among  the  substances  peculiar  to  the  intestinal  tract  we  find  bile, 
pancreatin,  and  other  enzymes  and  a  certain  water  percentage  and 
the  various  ingredients  of  food  materials  more  or  less  digested 
and  the  various  products  elaborated  by  the  different  species  and 
varieties  of  bacteria  present.  It  is  a  study  of  the  peculiarities  of 
the  intestinal  bacteria  which  has  suggested  the  technique  for 
their  isolation  and  their  quantitative  as  well  as  qualitative  esti- 
mation in  food  supplies,  as  can  be  seen  readily  from  a  study  of 
the  culture  media  and  cultural  methods  recommended.  To 
enter  into  any  fuller  discussion  of  the  work  done  by  American  and 
European  investigators  on  the  bacteria  which  are  normal  to  the 
intestinal  tract  or  which  may  occur  in  the  intestinal  tract  in 


94 


BACTERIOLOGICAL   METHODS 


disease,  is  not  practicable  but  we  desire  to  give  the  following 
tabulation  showing  the  relationship  between  the  different  species 
of  the  colon-typhoid  group  of  intestinal  bacteria  in  their  behavior 
with  dextrose  and  lactose  media. 


Bacteria  of  the  Colon-typhoid  Group 


Species 


Dextrose 


Gas  Formation 


Acid  Formation 


Lactose 


Gas  Formation  Acid  Formation 


B.  alcaligenes 

B.  typhi 

B.  dysenteric 

B.  enteritidis , 

Paratyphoid  group. 
Hog  cholera  bacillus 

B.  coli 


none 
none 
none 

active 
active 
active 

active 


none 
slight 
distinct 

strong 
strong 
strong 

strong 


none 
none 
none 

none 
none 
none 

active 


none 
none 
none 

slight 
slight 
slight 

strong 


There  are  numerous  other  distinguishing  characteristics  be- 
side those  indicated  in  the  above  tabulation,  as  agglutinating  phe- 
nomena and  behavior  with  other  special  culture  media.  It  is 
simply  desired  to  indicate  somewhat  more  specifically  the  lines 
of  research  which  were  necessary  to  determine  the  identity  of  the 
related  species  and  varieties  of  intestinal  bacteria. 

The  Bacillus  coli  was  isolated  as  early  as  1884  from  the  feces 
of  a  cholera  patient,  at  which  time  this  organism  was  supposed 
to  have  some  causal  relationship  to  cholera.  Later  it  was  proven 
that  this  bacillus  was  a  normal  inhabitant  of  the  intestinal  tract 
of  man  and  of  other  animals,  being  regularly  present  in  their 
excreta,  and  this  discovery  proved  of  the  highest  iriiportance  to 
sanitarians  as  the  presence  of  this  germ  in  water  supplies,  in  milk, 
in  mineral  waters,  etc.,  is  generally  regarded  as  evidence  of 
sewage  contamination.  The  colon  bacillus  has  been  found  in 
sewage  contamination,  river  water,  in  spring  water,  ice,  milk. 


QUALITATIVE   DETERMINATIONS  95 

cream,  butter,  buttermilk,  sour  milk  tablets,  mineral  waters, 
oysters,  clams,  flour,  oatmeal,  cornmeal,  cereals,  frozen  eggs,  dried 
nuts  and  fruit,  etc.  B.  coli  is  not  normally  present  in  sea  water 
and  its  occurrence  in  salt  water  shellfish  is  evidence  of  sewage 
contamination.  The  occurrence  and  general  distribution  of 
the  colon  bacilli  is  almost  in  direct  proportion  to  the  density  of 
the  population.  Animals  of  all  kinds  are  disseminators  of  colon 
bacilli,  particularly  the  larger  animals  as  the  horse,  cattle,  the 
dog  and  domestic  fowls.  Within  and  about  the  home,  the  house-fly 
and  the  stable-fly  are  the  chief  distributors  of  colon  bacilli.  We 
may  repeat  that  colon  bacilli  are  found  on  the  skin  of  persons, 
particularly  on  the  hands  and  under  the  finger  nails.  The  under- 
clothing worn  carries  these  bacilli  and  is  the  agent  instrumental 
in  distributing  them  over  the  exterior  of  the  body,  especially  in 
those  of  uncleanly  habits.  Water  in  which  the  hands  have  been 
rinsed  will  generally  yield  positive  colon  bacillus  tests.  It  is 
also  apparent  that  the  colon  bacillus  does  not  survive  for  a  great 
length  of  time  outside  of  its  natural  environment;  thus  sewage- 
contaminated  waters  purify  themselves  of  colon  bacilli  after  a 
time,  the  period  varying  with  the  temperature  and  the  amount 
of  organic  matter  present.  Thus  it  sometimes  happens  that  a 
water  supply  may  show  a  high  bacterial  count  and  yet  be  quite 
free  of  colon  bacilli.  As  a  rule,  however,  water  supplies  and 
substances  brought  in  contact  with  such  water  supplies  which 
show  a  high  general  bacterial  count,  will  also  show  a  comparatively 
high  count  in  colon  bacilli.  There  may  be  notable  exceptions  to 
this  rule.  A  water  supply,  or  other  liquid  substance,  may  show 
a  comparatively  low  bacterial  count  and  yet  yield  numerous 
colon  bacilli.  Such  an  occurrence  would  indicate  an  unusual 
source  of  extensive  sewage  contamination. 

From  the  foregoing  it  is  evident  that  the  sanitary  examination 
of  foods  and  drugs  resolves  itself  into  the  making  of  quantitative 
bacterial  counts,  as  already  fully  explained,  and  the  presumptive 
colon    bacillus    test,  with    an  occasional  test  for  other  specific 


96  BACTERIOLOGICAL   METHODS 

organisms,  as  will  be  explained  later.  It  is  also  evident  that 
the  isolation  or  the  identification  of  the  colon  bacillus  in  a  mixed 
contamination  as  all  sewage-contaminated  substances  are,  is 
not  as  simple  a  matter  as  might  appear  on  first  consideration. 
However,  the  presumptive  tests  for  the  presence  of  the  colon 
bacillus  in  definite  quantites  of  the  food  materials  or  liquids  used 
with,  or  associated  with,  certain  food  materials,  is  almost  uni- 
versally accepted  as  evidence  of  the  dangerous  contamination 
with  sewage.  It  is,  however,  quite  clear  that  health  officers 
should  not  adopt  hard  and  fast  rules  or  standards  for  the  con- 
demnation of  foods  because  of  such  evidence  of  sewage  con- 
tamination. Very  naturally,  the  standard  for  water  supplies 
will  not  apply  to  oysters  and  shellfish  generally  and  the  standard 
for  shellfish  will  not  be  practically  applicable  to  mineral  waters, 
etc.  With  substances  of  which  the  standard  or  quality  is  quite 
generally  based  upon  a  numerical  count,  as  for  example  milk, 
the  presumptive  colon  bacillus  test  need  not  be  applied,  unless 
it  is  to  be  carried  out  as  giving  corroborative  evidence  of  the 
sewage  contamination. 

One  of  the  first  important  duties  of  the  food  and  drugs  bac- 
teriologists will  be  for  them  to  get  together  and  agree  upon  uni- 
form methods  and  to  decide  upon  the  kinds  of  bacteriological 
examination  under  the  pure  food  and  drugs  act  to  which  the 
quantitative  as  well  as  the  qualitative  (presumptive  colon  bacillus 
test)  determinations  are  applicable,  in  harmony  with  our  present 
knowledge  of  food  bacteriology.  The  working  laboratory  methods 
adopted  must  be  practicable  and  must  be  carried  out  primarily 
as  a  better  protection  of  the  physical  well-being  of  the  consumer, 
incidentally  also  safeguarding  the  business  interests  of  the  con- 
scientious manufacturers.  The  following  suggestions  are  in- 
tended to  indicate  along  what  lines  the  practical  qualitative  work 
may  be  done  and  also  to  outline  certain  research  work  which  should 
be  carried  on  in  order  to  develop  the  working  methods  to  greater 
perfection  and  to  add  such  new  methods  as  may  prove  useful. 


SEWAGE   CONTAMINATION 


97 


10.  Evidence  of  Sewage  Contamination.     General  Methods. 


It  may  be  assumed  that  the  presence  of  any  or  all  of  the 
large  group  of  colon  bacilli  in  water  or  in  food  substances  is  indi- 
cative of  sewage  contamination  or  contamination  with  fecal 
matter.  The  colon  bacilli  are  aerobic,  nonsporeforming,  motile, 
short  and  produce  acid  and  gas  in  dex- 
trose and  lactose  media  and  develop 
best  at  a  comparatively  high  tempera- 
ture (37°  C.)-  A  practical  presumptive 
colon  bacillus  test  depends  upon  the 
characteristics  thus  indicated  and  is  car- 
ried out  as  follows: 

I.  Presumptive  Colon  Bacillus  Test. 
— Add  the  substances  to  be  tested 
(water,  sewage,  mineral  water,  shellfish 
liquor,  washings  from  vegetables,  etc.) 
in  o.oi  cc,  o.io  cc,  i  cc,  5  cc.  and  10 
cc.  quantities  (or  these  equivalents  in 
dilutions)  into  fermentation  tubes  hold- 
ing at  least  40  cc.  of  lactose  bile,  incu- 
bate at  37°  C.  and  look  for  the  forma- 
tion of  gas.  If  gas  formation  is  observed 
the  presence  of  colon  bacilli  may  be  sus- 
pected. If,  in  the  case  of  water  supplies 
for  example,  the  o.io  cc.  tubes  show  gas 
formation  then  it  may  be  reasonably  as- 
sumed that  colon  bacilli  are  present.     If 

two  out  of  five  of  such  tubes  give  positive  gas  reactions,  the 
test  may  be  considered  conclusive.  To  test  the  gas  formed, 
fill  the  tubes  showing  gas  formation  with  a  2 -per  cent,  solution 
of  sodic  hydrate,  hold  thumb  firmly  over  the  opening  of  the  fer- 
mentation tube  and  mix  contents  by  tilting  back  and  forth 
carefully.     The  volume  of  gas  absorbed  is  CO2  whereas  the  un- 


F I G .  25 . — Fermentation 
tube.  This  type  of  fermenta- 
tion  tube  is  especially  conven- 
ient for  making  the  gas  deter- 
mination with  the  colon  bacil- 
lus. Other  forms  of  fermenta- 
tion tube  may  be  used. — {Pitt- 
field.) 


gS  BACTERIOLOGICAL   METHODS 

absorbed  portion  is  supposedly  hydrogen.  The  colon  bacillus 
shows  a  gas  formation  of  3<3  hydrogen.  The  standard  time  of 
incubation  is  48  hr.,  but  if  colon  bacilli  are  abundant,  gas  forma- 
tion will  be  observed  in  the  tubes  carrying  the  larger  amounts  of 
the  inoculated  material  at  a  much  shorter  time,  occasionally  within 
a  few  hours.  Small  numbers  of  attenuated  colon  bacilli  may 
require  2  and  3  days  before  there  is  any  gas  formation 
noticeable.  In  this  connection  it  may  be  mentioned  that  the 
attenuated  colon  bacilli  indicate  remote  contamination,  as  all 
B.  coli  of  recent  contamination  develop  readily  in  lactose  bile. 


Fig.  26. — Bacillus  coli.     Superficial  colony  on  a  gelatin  plate  2  days  old  (X  21). 
— {McFarland  after  Heim.) 

This  constitutes  the  usual  presumptive  test  for  the  presence  of 
sewage  contamination.  Some  investigators,  however,  recommend 
that  the  test  be  supplemented  as  follows:  Plant  the  suitable 
quantities  or  dilutions  into  liver  broth  (in  test-tubes)  and  in- 
cubate at  37°  C.  for  about  12  hr.  and  then  transplant  these  cultures 
into  the  lactose  bile  as  above  explained.  The  liver  broth  en- 
richment medium  is  said  to  bring  out  the  attenuated  forms  of 
colon  bacilli.  In  routine  procedures  the  liver  broth  culturing  is 
usually  omitted  as  the  important  point  at  issue  is  the  determina- 
tion of  fairly  recent  contamination  with  sewage,  or  of  sewage 


COLON  BACILLUS   TEST  99 

contamination  in  large  amount,   and   the  lactose  bile  medium 
gives  conclusive  results  regarding  this. 

The  presumptive  colon  bacillus  test  is  to  be  supplemented 
further  as  follows:  Plate  suitable  dilutions  of  the  substances  to  be 
tested  for  sewage  contamination  (o.ooi  cc,  o.oi  cc,  o.io  cc, 
1. 00  cc.)  into  lactose  litmus  agar  Petri  dishes,  making  two  sets. 
Incubate  one  set  of  these  plate  cultures  at  20°  C.  and  the  other  at 
37°  C.  and  note  the  following: 

1.  The  relative  number  of  colonies  which  develop  at  the  two  temperatures. 

2.  The  number  of  acid-forming  colonies. 

The  time  of  incubation  at  the  lower  temperature  (20°  C.) 
should  be  3  days,  although  fairly  conclusive  results  may  be 
noted  at  the  end  of  the  second  day.  The  standard  time  of  incuba- 
tion at  the  higher  temperature  (37°  C.)  is  48  hr.,  although  certain 
results  may  be  noted  at  the  end  of  24  and  36  hr.  If  the  propor- 
tion of  high  temperature  colonies  is  high,  it  is  indicative  of  the 
presence  of  numerous  bacteria  derived  from  the  intestinal  tract. 
If  the  high  temperature  colonies  approximate  (numerically)  the 
low  temperature  colonies,  sewage  contamination  may  be  suspected. 
If  in  addition  many  of  the  high  temperature  colonies  show  pink 
or  vermiHon  (on  lactose-litmus  agar),  the  sewage  contamination 
is  practically  proven.  Both  the  colon  bacilli  and  the  sewage 
streptococci  show  pink  colonies  on  this  medium,  the  latter  being 
the  brighter,  more  vermilion  in  coloration.  This  coloration  is 
due  to  the  formation  of  acid  by  the  organisms  named  which  reacts 
with  the  litmus.  Examine  the  pink  colonies  under  the  micro- 
scope in  order  to  determine  which  are  the  colon  bacilli  and  which 
the  streptococci.  As  a  rule,  high  temperature  colonies  should  not 
exceed  i  :  100  as  compared  with  the  low  temperature  colonies.  It 
must  be  kept  in  mind  that  the  pink  colonies  may  turn  blue  within 
24  hr.  due  to  the  liberation  of  ammonia  and  amines.  Red 
colonies  indicate  lactose  fermentation  with  formation  of  acid,  but 
since  bacteria  other  than  the  colon  bacillus  form  acid  (notably 

8 


lOO 


BACTERIOLOGICAL  METHODS 


the  streptococci),  it  is  desirable  to  examine  such  colonies  micro- 
scopically and  to  inoculate  into  other  media  and  perhaps  to 
test  for  indol  formation,  in  order  to  obtain  satisfactory  proof  as 
to  whether  or  not  they  are  colon  bacilli. 

Neutral  red  (a  safranine  dye)  reduction  was  at  one  time  con- 
sidered a  very  important  check  test  for  the  colon  group.  Stokes, 
as  early  as  1904,  recommended  that  neutral  red  be  added  to  lactose 
broth  in  the  fermentation  tubes  which  contain  the  required  dilu- 


FlG. 


27. — B.  coli  showing  flagellae  stained  by  the  van  Ermengen  method  (X 
1000). — {MacNeal,  from  McFarland  after  Migula.) 


tions  of  the  liquids  to  be  examined.  30  to  50  per  cent,  gas  forma- 
tion in  the  closed  arms  of  the  tubes  and  the  change  of  the  neutral 
red  to  canary  yellow,  is  said  to  be  characteristic  for  the  colon 
group.  It  would  appear  that  the  majority  of  bacteriologists  are 
inclined  to  omit  the  neutral  red  test  as  being  of  little  value. 

The  production  of  indol  in  peptone  broth  or  solutions  is  another 
colon  bacillus  test  much  used  in  the  United  States.  Boehmes' 
modification  of  the  Ehrlich  method  is  now  generally  employed, 
made  as  follows:    Two  solutions  are  required. 


COLON  BACIXLT/S'  TES-J;        -  lOI 

Solution  No.  I 

Para-dimethyl-amido-benzaldehyde 4  parts 

Absolute  alcohol 380  parts 

Concentrated  HCl 80  parts 

Solution  No.  II 
Sat.  sol.  of  potassium  persulphate. 

The  indol  test  is  performed  as  follows :    Add  5  cc.  of  solution  I 
to  10  cc.  of  a  broth  culture  and  then  add  5  cc.  of  solution  II,  the 


Fig.  28. — Bacillus  of  typhoid  fever,  stained  by  Loeffler's  method  to  show  flagella 

(X  1000). — {Williams.) 

whole  being  then  shaken.     A  red  color  indicates  indol.     Some  of 
the  leading  bacteriologists  consider  this  a  very  valuable  test. 

The  so-called  hog  cholera  group  or  the  Gaertner  group  of  bacilli 
is  important  from  the  standpoint  of  the  food  bacteriologist.  The 
Gaertner  group  occupy  a  position  intermediate  between  the 
chemically  active  coli  group  and  the  chemically  inert  typhoid 
group  and  includes  the  following  important  species  or  rather  strains 
— the  Bacillus  enleritidis  strain  which  includes  many  of  the  bacteria 
isolated  in  cases  of  food  poisoning  and  some  of  the  B.  typhi  murium 


I02  '■\.-     .  .'•     '.  BACTE^IOLGplCAL   METHODS 

varieties,  as  B.  psittacosis,  and  B.  suipestifer  and  B.  paratyphosus  B. 
They  differ  from  the  typhoid  group  by  gas  formation  in  dextrose, 
and  from  the  colon  group  by  the  production  of  an  alkaline  reaction 
in  milk.  They  are  concerned  in  the  development  of  intestinal 
disturbances  such  as  dysentery  and  diarrhea.  No  practical 
routine  working  method  for  the  isolation  of  the  Gaertner  group 
has  as  yet  been  recommended.  Some  of  the  more  important 
cultural  characteristics  are  indicated  in  the  table  of  Bacteria  of 
the  Colon- typhoid  Group. 

Another  important  group  of  bacteria  from  the  standpoint  of 
the  food  bacteriologist  is  the  large  group  of  sewage  streptococci. 
They  occur  in  the  intestinal  tracts  of  many  animals.  There 
are  numerous  strains  of  this  group  and  they  are  somewhat  less 
widely  distributed  than  the  colon  group.  The  determination  of 
sewage  streptococci  adds  but  little  more  than  may  be  learned  from 
the  colon  test  and  for  this  reason  we  shall  not  enter  into  any  fuller 
discussion.  This  statement  also  applies  to  the  host  of  other  bac- 
teria and  related  organisms  which  are  more  or  less  constantly 
associated  with  sewage  and  sewage  contaminations. 

For  all  practical  purposes,  the  presumptive  colon  bacillus  test 
supplemented,  as  the  special  cases  may  require,  with  certain 
special  tests,  combined  with  the  quantitative  counts  by  the 
plating  method  (gelatin  media)  will  give  all  the  information  which 
is  necessary  to  judge  of  the  quality  of  certain  foods,  drinks  and 
medicamenta,  as  far  as  the  contamination  with  sewage  is  concerned. 
These  points  will  be  more  fully  discussed  under  special  heads. 

II.  Possible  Contamination  of  Foods  with  the  Typhoid  Bacillus 

Testing  food  substances  and  medicamenta  for  the  presence  of 
the  typhoid  bacillus  will  never  become  a  regular  routine  in  the 
food  laboratory.  On  occasion  it  will  become  an  incidental  pro- 
cedure and  must  therefore  receive  some  consideration.  To  under- 
stand the  special  significance  and  importance  of  this  organism 
as  a  possible  contaminator  of  foods,  it  is  necessary  to  enter  into 


TYPHOID  BACILLUS   CONTAMINATION 


103 


a  brief  statement  of  the  typhoid  fever  and  the  organism  which 
causes  this  disease.  The  primary  cause  of  typhoid  fever  is  the 
Bacillus  typhosus,  which  in  its  general  morphological  characteris- 
tics resembles  the  colon  bacillus,  differing  in  that  it  is  somewhat 
longer  and  more  actively  motile.  When  introduced  into  the 
intestinal  tract  of  man  it  multiplies  very  actively  and  produces 
the  symptoms  of  the  disease  known  as  typhoid  fever.  In  disease, 
therefore,  this  organism  grows  in  the  same  environment  as  the 
colon  bacillus,  excepting  that  the  temperature  (fever  temperature) 
is  higher.     After  recovery  from  the  disease,  the  germs  may  remain 


Fig. 


29. 


-Bacillus  typhosus,  72-hr.  gelatin  culture. — {Stitl,  after  Kolle  and 
Wassermann.) 


in  the  intestinal  tract  for  long  periods  of  time,  for  months  and  years. 
Furthermore,  those  who  have  never  had  the  disease  may  become 
infected  with  the  germs  and  carry  them  for  long  periods  of  time 
without  developing  the  disease.  Persons  infected  with  the  germs 
of  typhoid  fever  without  suffering  from  the  disease  are  known  as 
typhoid  carriers,  and  it  is  self-evident  that  they  may  cause  typhoid 
fever  in  those  with  whom  they  may  come  in  contact.  Numerous 
such  carriers  have  been  found  and  many  sporadic  cases  of  typhoid 
have  been  traced  to  such  source.  However,  the  majority  of 
typhoid  epidemics  are  traceable  to  foods  and  drinks  contaminated 


104  BACTERIOLOGICAL  METHODS 

with  the  intestinal  secretions  of  typhoid  patients.  The  subject 
of  typhoid  contamination  is  therefore  intimately  associated  with 
the  general  subject  of  sewage  contamination  or  contamination 
with  human  fecal  matter.  Very  naturally,  the  typhoid  bacillus 
is  far  less  common  than  the  colon  bacillus.  In  a  general  way  it 
may  be  stated  that  the  distribution  of  the  typhoid  bacillus  is  as 
wide  as  the  distribution  of  typhoid  contaminated  sewage.  As 
long  as  we  adhere  to  the  antiquated  and  highly  unsanitary  method 


Fig.  30. — B.  typhosus  from  gelatin  smear  preparation  stained  with  fuchsin  (X 

1000). — (MacNeal.) 

of  emptying  our  sewage  into  the  drinking-water  supplies  just  so 
long  will  we  continue  to  have  epidemics  of  typhoid  fever.  Numer- 
ous statistical  records  show  that  the  mortality  rate  from  typhoid 
fever  in  our  larger  cities  is  directly  proportional  to  the  filthiness 
of  the  drinking-water  supply.  House-flies  are  known  to  be  carriers 
of  typhoid  and  the  germs  have  been  isolated  from  vegetable  food 
materials,  from  oysters  and  other  shellfish,  from  milk,  etc. 

The  laboratory  procedure  in  the  examination  of  foods  and 
liquids  for  the  typhoid  bacillus  includes  the  isolation  and  identi- 


TEST   FOR   TYPHOID  BACILLUS 


105 


fication  of  the  germ.  The  proceedings  are  similar  to  those  out- 
lined for  the  colon  bacillus,  excepting  that  in  this  case  the  quan- 
titative factor  is  not  considered.  The  finding  of  a  single  typhoid 
fever  germ  in  a  mass  of  food  materials  is  sufficient  to  condemn  it. 
It  may  be  assumed  that  where  there  is  one  typhoid  bacillus  there 
are  more  in  the  same  vicinity  and  these  may  initiate  an  epidemic 
of  typhoid  fever. 

The  food  bacteriologist  may  be  called  upon  to  examine  food 
substances  for  the  presence  of  typhoid  contamination  (from  the 
feces  of  typhoid  patients  or  of  carriers)  in  instances  where  it  is 
known  that  food  has  been  exposed  to  typhoid  infection  or  where 
such  infection  is  merely  suspected.  The  isolation  from  foods  and 
the  positive  identification  of  the  typhoid  bacillus  is  by  no  means  a 
simple  matter.  It  is  necessary  to  make  use  of  special  cultural 
methods,  supplemented  by  the  agglutination  test,  etc.  The 
methods  tried  out  by  various  bacteriologists  are  too  numerous  to 
even  review  and  most  of  them  have  after  a  time  been  abandoned 
as  unsatisfactory.  The  following  tabulation  from  the  work  of 
Prescott  and  Wilson  indicates  some  of  the  more  practical  laboratory 
procedures  which  have  been  tried  with  more  or  less  success. 


Examination     of 

water  for  typhoid 

bacilli 


a.  By  filtration. 

b.  By  agglutination 

I. 

Physical    concen- 

Schuder's 

tration 

c.  By     chemical 

Fischer's 

^  Proc- 

precipitation 

Wilson's 

ess. 

MuUer's 

a.  Hoffman  and  Picker's  caffein  process 

2. 

Enrichment 

h.  Jackson's  lactose  bile. 
,  c.  Parietti's  carbol  broth. 
a.  Eisner's  gelatin  medium. 
h.  Endo's  medium. 

Isolation 

c.  Loeffler's  malachite  green  medium. 

3- 

d.  Drigalski-Conradi  agar. 

e.  Hiss's  medium. 

^  /.  Hesse's  medium. 

a.  Morphological  and  cultural  charac- 

4. 

Identification 

teristics. 

h.  Agglutination. 

io6 


BACTERIOLOGICAL   METHODS 


:lined*| 


Space  will  not  permit  discussing  the  methods  thus  outlined" 
nor  is  this  essential  for  the  present  purpose.  Those  interested 
are  referred  to  the  work  by  Prescott  and  Winslow,  Elements  oifl 
Water  Bacteriology  (1913),  which  contains  a  fairly  complete  digest  • 
of  the  methods.  Furthermore,  the  methods  adopted  must  be 
suited  to  the  special  cases  in  hand.  The  most  suitable  procedure 
for  isolating  the  Bacillus  typhosus  from  drinking  water  would 
not  be  practicably  applicable  in  the  examination  of  typhoid  con- 


??e~> 


Fig.  31. — B.  typhosus  from  an  agar  culture  6  hr.  old.  Highly  magnified 
(X  1000),  showing  the  flagellae  stained  by  the  Loeffler  method. — (McFarland  after 
MacNeal.) 

taminated  sewage  or  milk,  for  example.  For  the  time  being  there 
is  no  routine  laboratory  method  for  the  isolation  of  the  typhoid 
bacillus  and  we  must  content  ourselves  with  a  brief  consideration 
of  those  methods  which  will  in  all  probability  give  the  best 
results. 

It  is  of  the  highest  importance  that  the  food  bacteriologist 
should  search  out  typhoid  contaminated  foods  before  the  occur- 
rence of  an  epidemic.  In  fact,  if  such  work  is  not  undertaken 
until  cases  of  typhoid  have  developed,  the  bacteriological  find- 


TEST  FOR  TYPHOID  BACILLUS  107 

ings  are  often  wholly  negative,  because  of  the  long  incubation 
period  (14  days),  so  that  the  bacilli  may  all  have  disappeared 
from  the  sewage  or  water  between  the  time  of  the  infection  and 
the  manifestation  of  the  symptoms  of  the  disease.  Under  con- 
ditions favorable  to  the  typhoid  germs,  as  food  supply,  temperature, 
absence  of  sunlight,  etc.,  they  may  survive  for  several  months. 
It  is  generally  conceded  that  the  Bacillus  typhosus  is  quite  re- 
sistent  and  persistent.  According  to  Ravenel,  the  germ  survives 
for  several  months  and  longer  in  fecal  matter  deposited  in  snow 
which  when  carried  into  the  stream  supplying  a 
city  with  drinking  water  by  the  early  spring 
rains  caused  an  outbreak  of  typhoid. 

The  highly  objectionable  method  of  using 
human  excrement  for  fertilizing  the  soils  of 
truck  gardens,  as  practised  by  the  Chinese  and 
others,  may  lead  to  the  typhoid  contamination  ^  _.,„ 

of  the  vegetables  grown  in  such  gardens.  Wash-  trating  the  Widal 
ings  of  the  soil  and  of  the  vegetables  should  be  nomenon.  ^° U  p  p  e^r 
examined  for  typhoid  germs.  ^^^^  before  the  reac- 

rr.!       i-  „       .  1  1      ,   r         1       .     ,  tion.      Lower     half 

The  following  general  method  for  the  isola-     shows  clumping  of 

tion  of  the  typhoid  bacillus  is  suggested,  subject     lilJ^^j^ifilf/^^  ^^''' 

to  modification  to  suit  special  cases. 

1.  Concentration. — Run  from  i  to  lo,  and  more,  liters  of 
water  (as  from  well,  cistern,  stream,  water  tank,  etc.)  through  a 
clay  filter.  Just  before  all  of  the  water  has  passed  through  the 
filter,  shake  it  up  and  pour  into  a  suitable  centrifugal  tube  (the 
special  tube  already  described  will  answer  the  purpose  very  well) 
and  place  in  incubator  for  30  min.  at  a  temperature  of  37°  C. 
The  incubating  is  done  for  the  purpose  of  increasing  the  motility 
of  the  typhoid  bacilli. 

2.  Separation  by  Centrifugalization. — Take  tube  from  the 
incubator  and  centrifugalize  for  from  5  to  30  min.  at  a  high  speed. 
The  non-motile  bacteria  will  be  thrown  down  first,  while  the 


Io8  BACTERIOLOGICAL   METHODS 

highly  motile  Bacillus  typhosus  will  tend  to  remain  near  the  middle 
and  upper  parts  of  the  tube. 

3.  Cultural  Separation  on  Basis  of  Motility. — By  means  of  a 
sterile  pipette  take  up  the  upper  half  or  third  of  the  contents 
of  the  centrifugalized  tube  (2)  and  place  in  the  special  loop  tube 
with  phenol-broth  and  incubate  at  37°  C.  for  24  hr.,  or  longer  if 
necessary. 

4.  Plate  Cultures. — Take  up  several  platinum  loopfuls  from 
the  loop  tube  (the  opening  opposite  the  inoculated  end)  and 
plant  in  lactose-litmus-agar  (at  37°  C.)  and  note  the  character 
of  the  colonies  which  form.  Compare  with  the  colon  bacillus 
colonies.     Examine  colonies  microscopically. 

5.  Other  Cultural  Tests. — Test  for  absence  or  presence  of  gas 
formation.     Enrichment  in  liver  broth  may  be  tried,  etc. 

6.  Agglutination  Tests. — Two  methods  may  be  used.  The 
microscopical  and  the  macroscopical.  The  usual  routine  mi- 
croscopical method  is  carried  out  as  follows:  By  means  of  a 
clean  sterile  pipette  place  o.i  cc.  of  the  typhoid  serum  and  0.9 
cc.  of  physiological  salt  solution  (salt  is  necessary  to  bring  about 
agglutination)  in  a  clean  sterile  Syracuse  watch  crystal  and 
mix  thoroughly  by  means  of  a  clean  sterile  glass  rod.  This 
gives  a  serum  dilution  of  i-io.  Place  one  platinum  loopful  of  a 
24-hr.  bouillon  culture  of  the  typhoid  bacillus  on  a  clean  cover 
glass  and  add  one  loopful  of  the  mixture  from  the  Syracuse  watch 
glass.  This  gives  a  dilution  of  1-20.  Two  loopfuls  of  the 
culture  and  one  of  the  serum  mixture  gives  a  dilution  of  1-40. 
Three  loopfuls  of  culture  and  one  of  serum  mixture  gives  a  dilu- 
tion of  1-80.  Make  the  dilutions  one  at  a  time  and  place  the 
cover  glass  holding  them  (inverted)  on  a  vaselined  hollow  or 
concave  slide  and  examine  at  once  under  the  high  power,  con- 
tinuing the  observation  for  30  min.  if  necessary.  The  first 
change  noticeable  will  be  a  gradual  loss  of  motility,  followed 
by  a  clumping  of  the  now  non-motile  germs.  This  constitutes 
a   positive   agglutination   reaction.     Clumping   with    the   lower 


TYPHOID   AGGLUTINATION  TEST  IO9 

dilutions  (1-20,  1-40)  is  not  considered  characteristic  for  the 
typhoid  organism,  since  other  bacteria  may  also  produce  agglutina- 
tion with  the  typhoid  serum.  It  is,  however,  not  likely  that 
sera  will  agglutinate  other  than  the  specific  one  in  dilutions  as 
high  as  1-80.  Higher  dilutions  should  be  tried  on  the  principle 
that  the  positiveness  of  the  test  is  in  proportion  to  the  serum 
dilution  which  will  produce  clumping.  It  should  also  be  borne 
in  mind  that  the  agglutination  phenomena  are  more  marked  at 
the  body  temperature  (37°  C.)  and  that  in  the  case  of  the  typhoid 
serum,  the  paratyphoid  group  will  also  give  positive  results. 
In  reporting  on  the  agglutinating  phenomena  always  give  the 
dilution  and  the  time  factors.  The  novice  must  frequently  be 
reminded  that  all  manner  of  solutions  of  salts,  acids,  etc.,  will 
produce  agglutination  with  most  bacteria.  We  would  not 
recommend  the  use  of  the  blood-counting  pipette  (which  accom- 
panies the  hemacytometer)  for  making  the  dilutions  and  mixtures 
of  the  serum  and  the  bacterial  cultures,  as  is  advised  by  some 
investigators,  largely  because  of  the  danger  of  possible  infection 
in  sucking  up  the  quantities  of  bacteria,  and  also  because  this 
method  adds  nothing  to  the  value  of  the  results. 

For  the  so-called  macroscopical  method  or  precipitation  method, 
as  it  is  also  called,  small  test-tubes  are  used  in  which  the  suitable 
dilutions  of  the  serum  (with  normal  salt  solution)  and  the  bacterial 
cultures  are  mixed.  A  positive  reaction  is  indicated  by  flocculency 
and  the  deposition  of  a  slight  precipitate.  Dead  (formalized) 
typhoid  cultures  may  be  used.  The  method  in  general  use  in 
Germany  is  preferred,  a  description  of  which  may  be  found  in 
most  text-books  on  bacteriology.  Some  of  the  American  pharma- 
ceutical houses  (Parke,  Davis  &  Co.)  market  a  full  equipment 
for  making  the  macroscopic  agglutination  test  with  the  typhoid 
germ.  It  contains  full  directions  for  using  and  according  to  re- 
ports is  as  reliable  as  this  test  can  be  made  for  practical  purposes. 
It  need  hardly  be  stated  that  in  all  cases  it  is  desirable  to  make  a 
control  test  with  normal  salt  solution. 


no  BACTERIOLOGICAL   METHODS 

The  following  is  offered  by  way  of  fuller  explanation  of  some 
of  the  details  of  the  method  above  outlined  for  the  isolation  and 
identification  of  the  Bacillus  typhosus.  The  unusually  active 
motility  of  the  typhoid  germ  has  been  utilized  by  several  in- 
vestigators (Drigalski  and  Starkey)  as  a  means  for  separating 
it  from  less  highly  motile  forms.  Drigalski  allowed  from  5  to 
10  liters  of  the  suspected  water  to  stand  in  tall  milk  cans  for  i  or 
2  days  at  the  room  temperature,  after  which  he  plated  definite 
amounts  taken  from  the  surface  of  the  container  into  litmus - 
lactose-agar.  By  this  method  he  was  enabled  to  isolate  typhoid 
bacilli  from  several  contaminated  springs.  Starkey  used  glass  tubes 
bent  into  four  loops  which  after  being  filled  with  phenol  broth  were 
inoculated  at.  one  end  and  incubated  anaerobically  at  37°  C.  for 
24  hr.  The  more  actively  motile  typhoid  bacilli  found  their  way 
to  the  fourth  loop  from  which  they  were  isolated  by  plating.  The 
centrifugal  method  above  recommended  is  merely  an  adjunct  to 
the  methods  employed  by  Drigalski  and  Starkey.  The  non- 
motile  bacteria  are  thrown  down  first  and  in  a  very  short  period 
of  time  thus  being  an  advantage  over  the  Drigalski  method  in 
which  gravity  is  the  separating  force.  It  is  true  that  in  time  the 
motile  forms  would  also  be  thrown  down.  It  is  therefore  im- 
portant not  to  prolong  the  centrifugalizing  more  than  is  necessary. 
In  place  of  the  four-loop  Starkey  tube  we  would  suggest  the  use 
of  four  separate  tubes;  one  a  simple  U-tube  or  single-loop,  a  W-  or 
double-loop,  a  three-loop  and  a  four-loop  tube.  These  tubes,  after 
being  cleaned  and  sterilized  are  filled  with  phenol  broth  and  in- 
oculated at  one  end  at  the  same  time.  Incubate  at  37°  C.  or  even 
at  40°  C.  and  examine  loopfuls  taken  from  the  ends  opposite  the 
ends  inoculated  as  follows:  The  U-tube  at  the  end  of  6  hr., 
the  double-loop  tube  and  the  three-loop  tube  at  the  end  of  12  hr., 
the  three-loop  tube  (reexamination)  and  the  four-loop  tube  at  the 
end  of  24  hr.,  and  the  four-loop  tube  again  at  the  end  of  36  hr. 
if  necessary.  The  phenol  broth  and  the  higher  temperature  hinders 
the  growth  of  most  bacteria  without  checking  the  growth  of  the 


TEST  FOR  TYPHOID  BACILLUS 


III 


typhoid  germs.     These  conditions  will  enable  the  highly  motile 
Bacillus  typhosus  to  reach  the  more  remote  loops  first  where  they 
may  be  taken  out  by  means  of  the  platinum  loop  or  the  pipette. 
In  place  of  the  loop  tubes  above  described  and  which  can  be 


Fig.  2)3- — Loop  tubes  for  culturing  and  isolating  typhoid  bacilli  and  other 
motile  bacteria  as  explained  in  the  text,  i,  Single-loop  or  tj- tube;  2,  double-loop  or 
W-tube;  3,  three-loop  tube;  4,  four-loop  tube.  The  tubes  are  filled  with  phenol 
broth  or  other  desirable  media  and  inoculated  at  the  ends  marked  (a).  Material 
for  subculturing  and  for  microscopical  examination  is  taken  from  the  opposite  end 
(6),  at  varying  intervals  of  time. 

made  in  the  laboratory,  it  would  be  preferable  to  use  a  single  tube 
of  four  or  five  loops  provided  with  openings  at  each  of  the  upper 
turns  of  the  loops,  thus  making  five  or  six  openings  in  all,  from 
which  the  quantities  to  be  examined  and  plated  may  be  taken. 


112  BACTERIOLOGICAL  METHODS 

The  tubes  must  be  fastened  to  suitable  stands  or  supports  to  pre- 
vent, as  much  as  possible,  the  mechanical  mixing  of  the  contents 
after  the  inoculations  are  made.  It  is  perhaps  self-evident  that 
concentrates  or  high  contaminations  are  to  be  inoculated  into  the 
tubes.  The  tubes  should  be  large  enough  to  hold  at  least  50  to 
100  cc.  of  medium  and  suspected  water  in  equal  parts. 

12.  Possible  Contamination  of  Food  Substances  with  the  Cholera 

Bacillus 

In  the  United  States  the  contamination  of  foods  with  the 
cholera  vibrio  is  far  less  likely  than  the  contamination  with  the 
typhoid  fever  germ,  yet  it  is  a  possibility  to  be  reckoned  with. 


Fig,  34. — Spirillum  cholercB,  from  broth  culture,  stained  with  fuchsin  (X  1000). — 
{Stitt,  after  Kolle  and  Wassermann.) 

The  cholera  germ  is  found  in  the  feces  (but  not  in  the  urine)  of 
patients  and  in  the  feces  of  carriers,  in  which  regards  it  resembles 
the  typhoid  bacillus.  It  is  less  resistent  than  the  typhoid  organ- 
ism, disappearing  rapidly  from  the  stools,  usually  in  5  to  10 
days.  Under  certain  conditions  (as  in  fresh  water  supplies) 
the  infection  may  endure  for  longer  periods,  for  several  months 
and  more.    Like  the  typhoid  germ,  it  shows  some  marked  tend- 


THE   TEST   FOR   THE  BACILLUS   OF   CHOLERA  II3 

encies  to  locate  in  the  bile  duct  or  gall-gladder,  where  it  may  re- 
main dormant  for  a  long  period  of  time.  This  observation  has  led 
to  the  use  of  bile  as  an  enriching  medium  for  both  organisms. 

The  cholera  vibrio  work  in  the  food  and  drug  laboratory  may 
resolve  itself  into  the  isolation  of  the  germ  from  water  supplies, 
from  vegetables  and  possibly  from  feces  and  from  sewage,  and 
consists  in  the  use  of  special  culture  media,  special  cultural  methods, 
inoculation  methods  and  agglutination  tests.  It  is  interesting  to 
note  that  the  method  now  in  use  for  isolating  the  cholera  vibrio 
from  water  supplies  is  the  original 
Koch  method,  done  as  follows. 
Add  I  per  cent,  each  of  peptone 
and  salt  to  100  cc.  of  the  suspected 
water  and  incubate  at  38°  C. 
Examine  microscopically  at  inter- 
vals of  8,  12  and  18  hr.  As  soon 
as  curved  and  comma -shaped 
organisms  appear,  plate  on  agar 
and  make  such  additional  tests 
as  may  be  necessary  to  prove  the       .Fig.  35. — S.  cholerce  showing  invo- 

r  .  1        1     1  1      lution    forms  (X    1000). — (MacNeal, 

presence  of  the  cholera  germ,  such  after  VanEmengen.) 
as  the  nitroso  reaction,  agglutina- 
tion test,  Pfeiffer^s  phenomenon,  etc.  It  is  not  practical  to  enter 
into  a  fuller  discussion  of  the  subject.  More  complete  details  will 
be  found  in  the  works  on  bacteriology  and  in  bulletins  and  reports 
on  bacteriology  and  on  hygiene.  For  example,  the  U.  S.  Public 
Health  Service  has  worked  out  a  quick  routine  method  for  isolat- 
ing the  cholera  germ  from  feces,  used  in  the  U.  S.  Quarantine  Ser- 
vice and  at  the  quarantine  station  of  New  York,  as  reported  in 
the  Journ.  of  the  Am.  Pub.  Health  Association  (Dec,  191 1)  and 
a  condensed  summary  of  the  general  methods  may  be  found 
in  the  admirable  little  work  by  Stitt  (Practical  Bacteriology, 
Blood  Work  and  Parasitology,  1913).  Numerous  special  reports 
will  be  found  in  American  and  foreign  bacteriological  literature. 


114  BACTERIOLOGICAL   METHODS 


13.  Biological  Water  Analysis 


The  complete  biological  analysis  of  water  supplies  is,  as  a  rule, 
not  a  regular  routine  of  the  food  and  drugs  bacteriologist,  yet  he 
should  be  prepared  to  make  such  analysis  when  occasion  makes 
it  necessary.  The  food  bacteriologist  will  have  to  do  more  with 
the  analysis  of  sewage  contaminated  water  suppKes  and  with 
foods  and  other  substances  which  have  come  in  contact  with  such 
contamination. 

The  complete  biological  analysis  of  water  supplies  may  be  out- 
lined as  follows  the  fuller  details  of  which  may  be  found  in  special 
text-books,  bacteriological  journals  and  reports  on  water  analysis. 

Securing  the  sample. 
Bacteriological  examination. 

Quantitative. 

Qualitative;  the  presumptive  colon  bacillus  test. 
Algae;  significance  of. 

Diatoms. 

Desmids. 

Nostoc  and  oscillaria. 

Other  algae. 
Molds  and  spores;  significance  of. 
Ova  and  larvae  of  higher  parasites;  significance  of. 
Sand,  dirt,  etc. 

The  water  supply  of  a  city  or  community  should  be  watched 
at  all  times,  but  perhaps  more  particularly  in  the  early  spring  when 
the  melting  snows  and  the  heavy  rains  bring  in  materials  accumu- 
lated and  held  back  during  the  winter  months.  Furthermore,  the 
rise  in  temperature  encourages  the  rapid  multiplication  of  various 
organisms,  such  as  algae  and  bacteria.  In  late  summer  and  early 
fall  the  drinking  water  often  becomes  vitiated,  through  a  reduction 
in  supply,  perhaps  as  the  result  of  lack  of  rainfall.  In  the  early 
spring,  after  the  first  days  of  warm  weather,  the  water  supply  often 
becomes  murky  due  to  dirt  washed  in,  green  in  tint  due  to  the 
enormous  development  of  algae  and  generally  accompanied  by  a 
decidedly  disagreeable  odor  which  is  traceable  to  the  presence  of 


BIOLOGICAL   WATER  ANALYSIS  II5 

blue-green  algae  of  the  Nostoc  and  Oscillaria  groups.  Various 
more  or  less  futile  attempts  are  made  by  the  water  companies  to 
correct  these  conditions.  In  order  to  reduce  the  growth  of  algae  the 
reservoirs  are  roofed  over  (the  algae  requiring  sunlight  for  their 
development),  forgetting  that  while  one  evil  is  thus  in  a  measure 
corrected,  another  and  perhaps  greater,  is  encouraged  by  such  pro- 
cedure, namely,  the  growth  and  development  of  bacteria  which 
thrive  best  in  the  absence  of  sunlight.     Numerous  desmids,  di- 


FiG.  36. — S.  cholera  very  highly  magnified,  showing  flagellae. — (MacNeal,  from 
Kolle  and  SchUrmann.) 


atoms  and  blue-green  algae  in  drinking  water,  indicate  the  presence  of 
dead  and  decaying  organic  matter  in  comparatively  large  amount. 
Diatoms  are  especially  abundant  in  water  supplies  from  old 
wooden  tanks  and  wood-lined  reservoirs.  Nostoc  and  Oscillaria 
are  especially  abundant  in  water  supplies  fed  from  soil  drainage. 
Bacteria  are  present  in  all  soil  and  sewage  contaminated  waters. 
The  well  water  of  the  farms  may  be  contaminated  with  all  manner 
of  organisms,  such  as  sewage  organisms  and  disease  germs,  includ- 
9 


Il6  BACTERIOLOGICAL  METHODS 


Dthinfl 

c.      m\ 


ing  the  larvae  of  Nematodes  and  the  spores  of  fungi,  to  say  nothinj 
of  dead  and  decayed  animals  as  mice,  rats,  rabbits,  frogs,  etc. 

In  many  instances  the  contamination  (by  bacteria  and  algae)  of 
the  water  supplies  of  cities  and  towns  is  so  extensive  as  to  make 
rect  counting  easy.    We  hereby  give  the  report  of  the  microscopic^ 
examination  of  a  sample  of  water  from  a  Berkely  (Calif ornij 
reservoir,  analyzed  in  March,  191 2. 

Diatoms 1,500,000  per  cc. 

Desmids 860,000  per  cc. 

Oscillaria  filaments 125,000  per  cc. 

Bacteria 16,500,000  per  cc. 

Paramecia. 60,000  per  cc. 

Spores S,ooo  per  cc. 

Hyphae  of  fungi 460  per  cc. 

The  water  was  at  the  time  decidedly  greenish  in  tint  with  a  dis- 
agreeable odor,  due  to  the  numerous  algae  present.  Water  show- 
ing such  a  high  and  varied  biological  count  shows  surtace  seepage 
and  indicates  sewage  contamination  and  is  not  fit  for  drinking 
purposes,  yet  the  biologist  for  the  water  company  declared  it  good 
and  harmless.  The  only  interpretation  that  can  be  put  upon  a 
count  such  as  the  above  is  that  the  water  supply  is  dangerously 
contaminated.  Diatoms  and  desmids  feed  upon  dead  and  decay- 
ing vegetable  matter.  Oscillarias  occur  in  wet  soils  rich  in  humus. 
Paramecia  feed  upon  decaying  organic  matter.  The  molds  like- 
wise are  proof  of  the  decay  of  organic  matter,  animal  and  vegetable. 
In  all  cases  of  evidence  of  surface  seepage,  sewage  contamina- 
tion may  be  suspected  and  all  sewage  contaminated  drinking 
waters  are  a  menace  to  the  public  health. 

In  no  case  should  the  examination  of  concentrated  (1000  cc. 
reduced  to  10  cc.)  and  centrifugalized  sediment  be  omitted,  as  this 
will  perhaps  reveal  contaminations  which  might  be  overlooked  in 
the  direct  examination.  Nor  must  the  presumptive  colon  bacillus 
test  be  omitted  when  there  is  the  least  indication  that  sewage  con- 
tamination exists.     In  case  of  slight  but  suspicious  contaminations 


BIOLOGICAL  WATER  ANALYSIS  II7 

the  colon  bacillus  test  should  be  supplemented  by  the  plate  count 
and  the  examination  of  the  centrifugalized  sediment. 

Although  the  bacteriological  examination  of  water  supplies  is 
the  work  of  the  sanitarians,  the  food  bacteriologists  are  frequently 
called  upon  to  pass  judgment  on  the  potability  of  water  supplies. 
There  is  no  definite  numerical  standard  for  drinking  waters.  In 
the  United  States  the  presence  of  the  colon  bacillus  is  almost  wholly 
the  basis  for  condemnation,  it  being  assumed  that  if  bacteria  are 
present  in  great  numbers  the  colon  bacillus  is  also  generally  present. 
This  is,  however,  very  frequently  not  the  case.  Distilled  water  may 
contain  numerous  bacteria  without  any  colon  organisms.  Stag- 
nant waters  may  contain  bacteria  in  great  numbers  without  colon 
bacilli.  It  is  not  practicable  to  adopt  an  arbitrary  numerical 
limit  as  has  been  suggested  by  various  investigators.  Miquel 
(1891)  suggested  the  following  standards: 

10  bacteria  per  cc.  or  less Excessively  pure 

10-100  bacteria  per  cc Very  pure 

loo-iooo  bacteria  per  cc Pure 

1000-10,000  bacteria  per  cc Mediocre 

10,000-100,000  bacteria  per  cc Impure 

100,000  and  more  bacteria  per  cc Very  impure 

German  sanitarians  generally  recognize  a  limit  of  50  to  300  for 
drinking  water.  Dr.  Sternberg  of  the  Pubhc  Health  Service  (1892) 
suggested  that  less  than  100  bacteria  per  cc.  indicated  a  deep  source 
of  the  water  supply  and  uncontaminated  by  surface  drainage  and 
that  a  water  supply  with  500  bacteria  per  cc.  was  open  to  sus- 
picion and  that  1000  and  over  is  presumptive  indication  of  sewage 
contamination  or  of  surface  drainage.  It  is  quite  evident  that 
there  is  very  little  excuse  for  the  use  of  city  and  other  communal 
drinking  water  supplies  with  a  count  higher  than  5-10,000  per  cc, 
and  it  is  suggested  that  this  be  made  the  numerical  limit  for  drink- 
ing water  in  the  absence  of  or  irrespective  of  the  presence  of  the 
colon^bacillus. 

The  general  routine  for  making  the  tests  for  the  presence  of  the 


Il8  BACTERIOLOGICAL  METHODS 

colon  bacillus  has  already  been  explained.  It  is  suggested  that 
I  cc,  o.io  cc.  and  o.oi  cc.  quantities  of  the  water  be  run  into 
fermentation  tubes  with  lactose-bile  medium,  making  five  sets  of 
these  tube  cultures,  and  incubate  at  37°  C.  for  48  hr.,  noting  pos- 
sible gas  formation.  Gas  formation  indicates  sewage  contamina- 
tion. If  the  gas  is  formed  quickly,  in  6  to  1 2  hr.,  the  contamination 
is  probably  recent,  if  more  slowly,  24  to  36  hr.,  the  contamination 
is  probably  older.  Gas  in  the  o.oi  cc.  quantities  or  less,  indicates 
very  high  sewage  contamination,  gas  in  the  o.oi  to  o.io  cc.  quan- 
tities indicates  serious  contamination,  and  condemnation  of  the 
water  supply  for  drinking  purposes  may  be  based  on  the  presence 
of  gas  formation  in  two  out  of  three  tubes  containing  o.io  cc. 
quantities,  or  three  out  of  five  of  the  i  cc.  quantities,  also  tak- 
ing into  consideration  the  rate  of  gas  formation  and  the  numerical 
plate  count  as  well  as  the  findings  based  on  the  direct  microscop- 
ical examination.  In  brief,  condemnation  of  water  supplies  in- 
tended for  drinking  purposes  must  be  based  upon  the  judgment  of 
a  competent  sanitarian,  one  who  comprehends  the  significance  of  the 
findings  in  relation  to  the  source  of  the  water  supply  and  the  sources 
of  the  contaminations.  It  is  not  practicable  to  lay  down  hard  and 
fast  rules.  Each  case  must  be  considered  by  itself.  In  one  in- 
stance the  gas  formation  may  develop  in  0.3  cc.  quantities  (three 
out  of  five  tubes  containing  o.io  cc.  quantities)  or  even  in  o.io  cc. 
quantities  and  yet  the  water  may  be  considered  potable,  as  might 
be  the  case  in  deep  well  water  into  which  street  and  road  dust  is 
carried,  or  which  might  contain  surface  drainage  from  field  or 
garden.  Again  the  water  may  be  quite  unfit  for  drinking  pur- 
poses with  colon  bacilli  in  10  cc.  or  in  100  cc.  quantities,  as  might 
be  the  case  in  wells  or  springs  highly  contaminated  with  old  or 
much  weathered  sewage  contamination. 

14.  Bacteriological  Examination  of  Mineral  Waters        S 

The  bacteriological  analysis  of  bottled  waters  is  very  important 
because  it  is  an  efficient  means  of  ascertaining  the  conditions  at  the 


MINERAL   WATERS  IIQ 

bottling  establishments.  A  general  opinion  prevails  that  mineral 
waters  are  free  from  germs,  due  to  the  germ-destroying  properties 
of  the  mineral  salts  present.  This  is  not  the  case.  Many  mineral 
waters  from  contaminated  sources  or  from  unsanitary  bottling 
establishments  contain  bacteria  in  large  numbers,  300,000,000  per 
cc.  and  more.  Even  a  medicinal  water  composed  of  concentrated 
ocean  water  (Magpotine)  gave  a  count  of  10,000  bacteria  per  cc. 
The  Bureau  of  Chemistry  has  found  mineral  waters  contaminated 
with  sewage.  Often  the  contamination  is  traceable  to  the  inade- 
quate cleansing  and  sterilizing  of  used  bottles  and  to  the  dirty 
hands  of  those  employed  in  the  factory. 

The  bacteriological  examination  of  mineral  waters  consists  in 
making  the  presumptive  colon  bacillus  test  and  in  making  bacte- 
rial counts  by  the  plating  method.  It  is,  however,  also  desirable 
to  make  direct  microscopical  examinations,  including  quantitative 
cytometric  counts  of  concentrates  (i  liter  quantities  reduced  to 
10  cc.)^and  of  centrifugalized  samples,  as  already  explained.  This 
will  give  information  regarding  factory  conditions  which  could  not 
be  ascertained  by  the  usual  plating  methods. 

In  the  case  of  bottled  mineral  waters,  the  securing,  handling  and 
shipping  of  samples  is  a  very  simple  matter  as  no  extra  precautions 
and  care  are  necessary.  In  the  case  of  water  from  mineral  springs 
or  artificial  w^aters  in  bulk,  the  securing  of  samples  for  examination 
must  be  done  carefully* to  guard  against  outside  contamination. 
Containers  for  samples  must  be  clean  and  sterile  and  as  soon  as  the 
sample  is  taken  the  container  must  be  closed  with  a  sterilized  cork 
or  other  suitable  stopper,  sealed  and  taken  to  the  laboratory  by  the 
shortest  route  for  immediate  examination.  If  the  samples  are  to 
be  transported  long  distances  or  if  for  any  other  reason,  the 
examinations  must  be  postponed  for  from  6  hr.  to  several  days,  the 
sample  must  be  kept  on  ice  during  the  entire  period. 

Mineral  waters  are  or  should  be  quite  free  from  bacteria  and 
other  contaminating  organisms.  As  yet  no  standards  have  been 
adopted  as  to  the  maximum  number  of  bacteria  and  other  organ- 


I20  BACTERIOLOGICAL  METHODS 

isms  permissible.     The  only  quality  test  made  by  the  Bureau  of 
Chemistry  is  for  the  colon  bacillus. 

15.  The  Microscopical  and  Bacteriological  Examination  of  Milk 

It  is  not  practicable  to  enter  into  a  discussion  of  the  dairying 
industry  or  the  multitudinous  factors  which  cause  modification  of 
the  quality  of  cow's  milk.  These  are  matters  which  concern  the 
food  bacteriologist  but  little.  Bovine  diseases,  inclusive  of  tuber- 
culosis, must  be  left  to  the  veterinarian  and  the  making  of  dairy 
products  concern  the  manufacturer  primarily.  By  this  it  is,  how- 
ever, not  intended  to  imply  that  the  food  bacteriologist  need  not 
have  intimate  knowledge  of  cattle  diseases  and  of  dairying  meth- 
ods. Not  only  should  he  be  well  informed  regarding  these  things 
but  he  should  be  qualified  to  examine  cattle  for  diseases,  tubercu- 
losis in  particular,  and  should  be  prepared  to  examine  and  report 
upon  the  sanitary  conditions,  equipment  and  the  moderness  of  dai- 
rying establishments.  However,  the  chief  efforts  of  the  food  bac- 
teriologist are  devoted  to  the  examination  of  the  milk  and  dairying 
products  as  they  appear  upon  the  market. 

For  the  present  purpose  it  will  suffice  to  give  a  mere  outline  of 
the  methods  of  examining  and  testing  milk  microscopically  and 
bacteriologically.  The  report  of  the  analysis  should  comprise  the 
following. 

Securing  the  sample. 
Sealing  the  sample. 

Keeping  sample  on  ice  until  ready  for  examination. 
Examining  the  sample. 
Direct  examination. 
Determining  the  fat  content  by  the  microscopical  method. 
Quantitative  determination  of 
Bacteria. 
Epithelial  cells. 
Blood  corpuscles. 
Pus  cells  and  leucocytes. 
Plate  cultures. 

Presumptive  colon  bacillus  test. 
Numerical  count. 


MILK 


121 


Milk  may  be  described  as  a  uniform  suspension  of  fat  globules 
in  an  aqueous  solution  of  milk-sugar  and  casein.  The  fat  globules 
represent  the  so-called  butter  fat  of  the  milk.  They  are  fairly  uni- 
form in  size,  very  uniformly  distributed  and  under  ordinary  con- 
ditions do  not  tend  to  coalesce  or  clump.  Pasteurization  and 
boiling  the  milk  does  cause  some  of  the  globules  to  unite  or  rather 
to  form  aggregates  but  even  in  such  cases  it  is  possible  to  recognize 
the  individual  globules. 

On  mounting  a  droplet  of  diluted  milk  (1-150  to  1-200)  on  the 
hemacytometer  it  will  be  found  that  the  fat  globules  soon  rise  to 


Fig.  37. — Milk  fat  globules.  Larger  field  as  they  appear  under  the  low  power  of 
the  compound  microscope  (X  80),  globules  in  the  corner  circle  as  they  appear  under 
the  high  power  (X  500). — {Hunter,  after  S.  M.  Babcock.) 

the  top  while  the  heavier  particles,  such  as  bacteria  and  body  cells, 
sink  to  the  bottom  of  the  cell,  thus  separating  these  elements  auto- 
matically, and  making  the  counting  of  globules  and  bacteria  pos- 
sible in  the  same  mount  by  simply  focusing  sharply  upon  the  fat 
globules  or  upon  the  bacteria  as  may  be  desired.  Some  difficulty 
in  making  the  counts  is  caused  by  the  fact  that  the  oil  globules  are 
out  of  focus  when  the  rulings  are  in  focus,  making  a  constant  shift- 
ing of  focus  from  fat  globule  to  lines  and  vice  versa  from  lines  to  fat 
globule,  necessary.  Not  only  is  this  annoying  but  it  makes  accu- 
rate counting  difficult.     This  difficulty  can  be  overcome  by  com- 


122  BACTERIOLOGICAL   METHODS 

billing  the  use  of  an  eye-piece  micrometer  scale  with  that  of  the 
hemacytometer,  and  it  is  suggested  that  such  a  combination  be 
used,  not  only  for  milk  examination,  but  also  for  making  many  of 
the  cytometric  counts  of  food  products. 

A  practical  method  for  determining  the  fat  content  of  milk  by 
means  of  the  compound  microscope  was  worked  out  in  the  bacterio- 
logical laboratory  of  the  California  College  of  Pharmacy.  The  pro- 
cedure is  as  follows:  Make  dilutions  of  the  milk  from  1-150  to 
1-200,  using  distilled  water  or  normal  salt  solution  (0.6  per  cent.) 
and  count  the  fat  globules  by  means  of  the  hemacytometer  or  the 
special  counter  above  suggested.  Numerous  counts  made  have 
shown  that  578,100,000  fat  globules  in  i  cc.  of  milk  corresponds  to 
I  per  cent,  of  butter  fat.  This  number  was  obtained  by  comparing 
the  fat  globule  count  with  the  fat  determination  by  the  standard 
chemical  method  (combined  with  the  use  of  the  centrifugal 
machine).  The  following  are  a  few  comparisons  as  they  were  ob- 
tained in  the  laboratories  of  the  California  College  of  Pharmacy. 

1,383,000,000  fat  globules  per  cc.  corresponded  to  2.30  per  cent,  of  butter  fat. 
933,000,000  fat  globules  per  cc.  corresponded  to  i  .60  per  cent,  of  butter  fat. 
566,000,000  fat  globules  per  cc.  corresponded  to  i .  10  per  cent,  of  butter  fat. 
470,000,000  fat  globules  per  cc.  corresponded  to  0.80  per  cent,  of  butter  fat. 

Dividing  the  sum  total  of  the  several  counts  of  fat  globules 
made  by  the  sum  total  of  the  corresponding  percentages  of  butter 
fat,  gives  578,100,000  the  average  number  of  globules  in  i  cc.  of 
milk  corresponding  to  i  per  cent,  of  butter  fat.  From  this  it  will 
be  seen  that  in  round  numbers,  2,000,000,000  fat  globules  per  cc. 
represent  a  fair  quality  of  milk,  that  is,  milk  having  somewhat  over 
3.50  per  cent,  of  butter  fat.  According  to  comparative  tests  made, 
the  microscopical  method  is  fully  as  accurate  and  rehable  as  the 
chemical  methods.  The  microscopical  method  is  not  recom- 
mended for  routine  procedure  in  dairying  establishments  but  it 
is  certainly  a  most  valuable  adjunct  to  the  food  laboratory 
methods.  It  could  at  all  times  be  employed  as  a  substitute  for 
the  chemical  fat  determination  if  for  any  reason  the  latter  method 


BACTERIOLOGICAL   STANDARDS   FOR   MILK 


123 


is  not  applicable.  Thus,  it  can  be  ascertained  microscopically 
whether  or  not  water  has  been  added  to  the  milk  or  if  it  is  full 
milk,  half  milk  or  skimmed  milk. 

The  bacteriological  standardization  of  milk  has  received  much 
attention  within  recent  years  and  all  civilized  countries  have 
adopted  certain  numerical  limits  of  bacteria  permissible  in  whole- 
some milk.  Unfortunately,  however,  there  is  very  little  uniformity 
regarding  these  numerical  limits  in  different  countries  or  in  differ- 
ent parts  of  the  same  country.     In  some  cities  and  communities 


Fig.  38. — Milk  fat  globules  very  highly  magnified  (X  1000).     A  group  of  lactic 
acid  bacteria  at  the  left. — {Hunter.) 


there  are  two  standards,  a  summer  or  low  (numerical  limit  higher) 
standard  and  a  winter  or  high  (numerical  limit  lower)  standard. 
The  terms  summer  and  winter  are,  however,  misleading  in  certain 
areas  of  the  United  States  and,  for  regulatory  purposes,  it  would  be 
better  to  base  the  standards  on  a  temperature  differential,  irre- 
spective of  season,  combining  this  with  a  sliding  scale  of  bacterial 
count.  Under  such  a  plan  the  Southern  States,  including  the 
immediate  Pacific  Coast  region,  would  be  under  a  single  standard, 
namely,  the  lower  or  so-called  summer  standard.  The  rest  of  the 
United  States  would  have  both  standards. 


124 


BACTERIOLOGICAL  METHODS 


The  following  is  a  tentative  standard  based  upon  the  tempera- 
ture differential  as  above  suggested. 


Number  of  Bacteria  per  Gc. 

Standards 

Ordinary- 
Milk 

Certified 
Milk 

Inspected 
Milk 

Cream  (Fresji 
or  Unripened)! 

Temp,  from  lowest  to  60°  F. 
Winter  standard 

30,000  to 

50,000 

3,000  to 
8,000 

12,000  to 

15,000 

30,000  to 
5,000,000 

Temp,  from  6o°F.  to  highest. 
Summer  standard 

50,000  to 
100,000 

8,000  to 
15,000 

15,000  to 
30,000 

5,000,000  to 

150,000,000 

It  is  not  practicable  to  fix  a  numerical  bacterial  limit  for  creams. 
Tests  made  show  that  the  count  varies  within  wide  limits,  even  in 
cream  from  milk  which  has  been  kept  under  the  most  favorable 
sanitary  conditions  and  surroundings.  Fresh  creams,  that  is,  the 
cream  removed  from  the  milk  as  soon  as  formed,  usually  within 
24  hr.  after  the  milk  is  drawn,  contains  comparatively  fewer  bac- 
teria than  the  cream  which  has  been  set  aside  to  ripen.  The 
ripening  process  is  far  from  objectionable,  in  fact  it  is  encouraged 
and  regulated  in  the  well-conducted  dairying  establishments  in 
order  to  develop  the  desirable  butter  flavor.  Most  of  these 
flavoring  lactic  acid  bacteria  are  removed  in  the  process  of  butter 
making,  being  drawn  away  and  worked  out  with  the  buttermilk, 
only  comparatively  few  remaining  in  the  butter  itself. 

Taking  milk  samples  is  not  unlike  water  sampling.  Milk 
should  be  examined  not  later  than  6  hr.  after  being  drawn.  If  it 
cannot  be  examined  within  that  time  it  must  be  kept  on  ice  but 
in  no  case  should  the  examination  be  made  later  than  1 2  hr.  after 
the  milk  was  drawn. 

Body  cell  counts  should  not  be  omitted  and  proper  judgment 
should  be  exercised  in  interpreting  the  findings.     Body  cell  counts 

1  Ripened  cream  contains  numerous  lactic  acid  bacilli,  300,000,000  per  cc,  and 
even  more. 


BODY  CELLS   IN  MILK  125 

give  most  valuable  information  regarding  the  health  condition 
of  the  cows  and  will  serve  to  indicate  the  danger  point  as  to  the 
usability  of  the  milk.  It  is  not  practicable  to  give  exact  numerical 
limits  at  the  present  time.  Further  investigation  is  necessary  to 
this  end.  However,  the  following  suggestions  will  be  of  great 
value  to  the  analyst  in  arriving  at  a  better  estimate  of  the  quality 
of  the  milk  under  examination. 

Epithelial  cells  few  (looo  per  cc),  of  no  significance. 

Epithelial  cells  many  (5,000,000  per  cc.  and  more),  indicates  some  irritation  or  seri- 
ous inflammatory  condition  of  udder  or  in  milk  ducts. 

Epithelial  cells  many  with  some  pus  cells,  danger.  The  diseased  animal  should 
be  found  and  remov^ed  from  the  herd. 

Pus  cells  few,  indicates  some  slight  abscess  formation  which  should  be  treated  if 
possible. 

Pus  cells  many  (5,000,000  per  cc.  or  more)  indicates  danger.  The  diseased 
animal  should  be  removed  from  the  herd. 

Blood  corpuscles  few,  no  special  significance.  Probably  due  to  some  slight  injury 
resulting  in  capillary  hemorrhage. 

Blood  corpuscles  many.  Indicates  some  mechanical  injury  which  requires 
attention. 

For  practical  purposes  it  is  not  advisable  to  attempt  to  dis- 
tinguish between  leucocytes  and  pus  corpuscles.  Numerous  leuco- 
cytes indicate  some  serious  inflammatory  condition  while  numer- 
ous pus  cells  indicates  abscess  formation  perhaps  following  a 
more  severe  inflammation. 

Various  methods  have  been  submitted  for  making  the  body 
cell  counts.  That  by  Prescott  and  Breed  is  perhaps  the  simplest 
and  also  the  most  practical.  It  is  carried  out  as  follows.  Spread 
o.oi  cc.  of  the  milk  on  a  glass  slide^  over  an  area  of  i  sq.  cm., 
evaporating  the  milk  to  dryness  using  moderate  heat.  Next 
dissolve  out  the  butter  fat  by  means  of  xylol,  fix  with  alcohol, 
again  dry,  and  stain  with  methylene  blue.  Decolorize  partially 
with  alcohol  and  examine  under  the  compound  microscope.  The 
body  cells  in  the  entire  area  of  the  mount  are  counted  and  the 

*  The  ruled  slide  elsewhere  described  {D,  Fig.  5)  will  be  found  very  useful  for 
counting  body  cells  in  definite  quantities  of  the  milk. 


126 


BACTERIOLOGICAL  METHODS 


entire  number  found  multiplied  by  loo  gives  the  number  of  body 
cells  per  cc.  of  the  milk.  Prescott  and  Breed  have  examined 
numerous  milk  samples  and  declare  that  the  average  number  of 
body  cells  is  1,500,000  per  cc.  and  that  a  count  as  low  as  100,000 
per  cc.  is  uncommon. 

Little  can  be  said  regarding  the  microscopical  and  bacteriolog- 
ical examination  of  butter,  cheese,  cream  and  other  factory  products. 


Fig.  39. — Unglazed  porcelain  filters.     Chamberland  system;  A,  without  pressure; 
Bf  fitted  to  main  water  supply;  C,  section  of  a  porous  porcelain  filter. 

There  are  no  bacterial  standards  and  the  laboratory  work  is  very 
largely  limited  to  the  detection  of  adulterants  such  as  excess  of 
salt,  of  water  and  the  presence  of  lard  and  oleomargarine  in  but- 
ter, fillers  in  cream  and  in  ice  cream,  etc. 

The  following  simple  tests  will  be  found  useful  in  the  labora- 
tory: 

I.  Spoon  Test  for  Oleomargarine  and  Renovated  Butter. — Melt  a  small  piece 
of  the  suspected  butter  in  a  tablespoon  or  small  dish,  using  a  small  flame.  Stir  the 
melting  substance  with  a  small  piece  of  wood  such  as  a  tooth-pick  or  match.     At  a 


MILK  BACTERIA  1 27 

brisk  boil,  oleomargarine  and  renovated  butter  will  sputter  very  briskly  and  noisily 
without  foaming.  Genuine  butter  boils  less  noisily  and  with  abundant  foam 
formation. 

2.  Fat  Cohesion  Test — Fill  a  medium  beaker  about  half  full  of  sweet  milk  (pref- 
erably skimmed)  and  heat  to  within  near  the  boiling  point.  Add  about  5  grams  of 
the  sample  and  stir  until  completely  melted.  Remove  from  the  fire  and  place  beaker 
in  ice  water.  When  the  fat  begins  to  congeal,  stir  with  a  small  piece  of  stick.  Fat  or 
oleomargarine  will  collect  in  one  mass  or  lump  at  the  end  of  the  stick,  whereas  pure 
butter  granulates  and  will  not  adhere  to  the  stick.  This  test  is  rot  applicable  to 
renovated  butter  which  behaves  like  unrenovated  or  fresh  butter. 

As  is  generally  known,  milk  is  an  excellent  culture  medium  for 
a  great  variety  of  bacteria.  For  a  time  after  the  milk  is  drawn, 
bacterial  development  is  checked  by  the  bacterolytic  properties 
which  all  fresh  milk  is  said  to  possess.  These  lysins,  however, 
gradually  grow  less  and  less  until  there  is  no  longer  any  evidence 
of  their  existence. 

Milk  bacteria  may  be  grouped  into  the  acid  formers,  digesting 
bacteria  and  those  which  appear  to  have  but  little  effect  on  the 
appearance  of  the  milk.  The  acid-forming  group  is  a  large  one  and 
includes  the  true  lactic  acid  bacteria  which  are  carried  into  the 
milk  from  stable  dust  and  other  dirt  in  and  about  the  stables  and 
elsewhere.  The  initial  bacterial  changes  in  the  milk  are,  however, 
not  produced  by  the  acid  formers,  but  rather  by  those  bacteria 
which  decompose  proteids,  to  which  belong  the  B.  suhtilis  and  its 
aerobic  allies.  Streptococcus  acidi  lactici  ferments  both  proteids 
and  lactose  as  does  also  B.  coli  communis  and  some  of  its  allies. 
In  a  short  time,  however,  the  true  lactic  acid  bacteria  multiply  in 
such  large  numbers  as  to  crowed  out  or  almost  completely  check  the 
development  of  the  other  species.  They  transform  the  lactose 
into  lactic  acid.  On  longer  exposure,  Oidium  lactis  enters  from  the 
atmosphere  which  fungus  begins  to  decompose  the  lactic  acid 
and  some  of  the  remaining  proteids,  having  the  effect  of  lowering 
the  acidity  which  again  encourages  the  renewed  multiplication 
of  the  lactic  acid  group.  This  alternating  preponderance  of 
lactic  acid  bacteria  and  higher  fungi  continues  until  the  proteids 
and  the  milk  sugar  are  almost  completely  used  up.     Butyric  acid 


128  BACTERIOLOGICAL  METHODS 

bacteria  may  enter  the  milk  causing  the  very  characteristic  fer- 
mentation changes  resulting  in  the  formation  and  liberation  of  bu- 
tyric and  propionic  acids  from  the  splitting  of  lactose.  Butyric  acid 
milk  has  a  very  disagreeable  odor.  Various  bacteria  cause  dis- 
eases of  milk  as  blue  milk  and  ropy  milk. 

In  some  American  cities  the  routine  examination  of  milk  for 
B.  coli  is  regularly  adopted.  The  results  in  Baltimore  have  shown 
the  presence  of  this  bacillus  in  25  per  cent,  of  o.ooi  cc.  quantities 
of  the  milk  in  the  winter  time  and  75  per  cent,  during  the  summer. 
It  would  appear  that  three  positive  tests  out  of  a  total  of  five 
from  O.OOI  cc.  quantities  of  milk,  would  indicate  the  danger  point 
as  to  quality.  For  making  plate  counts  of  milk  bacteria,  lactose- 
litmus-agar  should  be  used  in  order  to  differentiate  between 
acid  formers  and  non-acid  formers. 

In  most  communities  the  milk  streptococci  are  considered 
objectionable,  as  they  belong  to  the  group  of  pus-forming  organ- 
isms. It  is  frequently  found  that  a  high  streptococcus  count  goes 
with  a  high  leucocyte  count  and  the  two  are  corroborative  of  the 
existence  of  some  severe  inflammatory  condition  of  the  udder  or 
milk  ducts.  There  is  fairly  conclusive  evidence  that  the  hemo- 
lytic milk  streptococci  are  frequently  causative  of  more  or  less 
severe  and  even  fatal  intestinal  diseases  among  children,  especially 
during  the  hot  summer  weather.  It  is  also  fairly  well  proven  that 
some  of  the  throat  and  mouth  infections  of  children  are  traceable 
to  the  staphylococci  and  streptococci  of  milk.  The  problem  of 
tuberculous  milk  is  of  lesser  importance  to  the  food  bacteriologist 
because  the  health  authorities  of  the  land  have  this  matter  under 
jurisdiction.  It  is  criminally  unlawful  to  market  milk  from  tuber- 
cular cows.  Ravenel  states  that  approximately  20  per  cent,  of 
the  clinical  cases  of  tuberculosis  are  of  the  bovine  type  and  milk 
from  tuberculous  cows  continues  to  be  a  very  serious  menace  to 
the  public  health.  It  would  be  of  the  greatest  value  if  some  simple 
and  practical  micro-chemical  laboratory  test  for  tuberculous  milk 
could  be  worked  out.     We  would  suggest  this  as  one  of  the  very 


HYDROGEN  DIOXIDE   MILK  TEST  1 29 

important  problems  to  be  undertaken.  It  is  evident  that  the  con- 
trol exercised  by  the  health  authorities,  while  it  has  accomplished 
much,  is  not  sufficiently  stringent  or  far-reaching  to  stamp  out 
tuberculosis   in   cows. 

A  milk  test  much  used  in  Holland  and  other  European  countries 
is  to  ascertain  the  amount  of  gas  formation  in  a  unit  of  time,  in  a 
fermentation  tube  containing  a  mixture  of  definite  quantities  of 
milk  and  hydrogen  dioxide.  The  amount  of  gas  liberated  is  di- 
rectly proportional  to  the  amount  of  organic  matter  (bacteria, 
body  cells  and  other  organic  impurities)  present.  Tests  made  in 
the  laboratories  of  the  California  College  of  Pharmacy  and  in-  the 
laboratories  of  the  San  Francisco  Board  of  Health  would  indicate 
that  the  method  gives  uniform  results  and  that  such  a  method 
would  prove  a  most  valuable  addition  to  the  routine  milk  examina- 
tion, serving  as  a  check  and  confirmation  of  the  bacterial  and  body 
cell  counts.  In  order  that  the  test  may  yield  uniform  results  in  all 
laboratories,  a  uniform  method  of  procedure  must  be  adopted. 
The  following  tentative  method  is  suggested. 

A  standard  10  percent,  volume  (of  available  oxygen)  solution  of 
hydrogen  dioxide  should  be  used.  The  peroxide  should  be  standard- 
ized to  the  specified  quality.  For  determining  the  valuation  of  the 
peroxide  we  would  recommend  the  Planes  colorimetric  test,  made 
as  follows.  Dilute  the  dioxide  to  be  tested  with  nine  parts  distilled, 
water.  To  5  cc.  of  this  solution  (i-io)  add  3  cc.  of  a  10  per  cent 
solution  of  potassium  iodide  and  i  cc.  of  8  per  cent,  sulphuric  acid, 
in  a  standard  test-tube.  The  color  produced  is  matched  against  a 
n/io  iodine  solution  in  a  second  standard  test-tube.  1.8  cc.  of 
the  standard  solution  is  equivalent  to  i  cc.  of  oxygen. 

Into  graduated  fermentation  tubes  with  slender  arms,  having  a 
capacity  of  25  cc,  run  10  cc.  of  milk  and  10  cc.  of  the  standard 
hydrogen  dioxide,  mix  well  in  the  bulb  and  at  once  run  into  the 
arm,  excluding  all  air  from  the  upper  end  of  tube.  Set  aside  in  the 
incubator  for  i  hr,  at  a  temperature  of  20°  C.  and  record  the 
amount  of  gas  formed  at  the  end  of  this  period. 


130 


BACTERIOLOGICAL  METHODS 


Fig.  40. — Streptococcus  {Staphylococcus)  pyogenes  and  5.  aureus.  There  arc 
three  principal  species  of  Streptococci  {S.  pyogenes  albus,  S.  pyogenes  aureus  and  S 
pyogenes  citreus),  similar  in  form  and  appearance,  concerned  in  pus  formation,  as  ir 
wound  infection.     These  organisms  are  very  widely  distributed  in  soil  and  air. 

Note  the  chain  form' arrangement  of  the  cocci  in  ^.  ^  is  a  smear  preparation. — 
( Stitt  (A )  and  Pittfield  {B).) 


TEST  FOR   WATERED   MILK  I31 

The  following  quick  and  simple  test  is  recommended  to  dis- 
tinguish between  raw  and  boiled  milk! 

Reagent 

Methylene  blue  (alcoholic) 5  cc. 

Formaldehyde  (40  per  cent.) 5  cc. 

Water  (distilled) 190  cc. 

Add  I  cc.  of  this  reagent  to  20  cc.  of  the  milk  and  heat  for  10 
min.   at  40^-45°  C.     Raw  milk  is  decolorized 
while  boiled  milk  retains  the  blue  coloration. 
This  test  should  in  all  cases  be  checked  by  the 
microscopical  examination.     Boiling  the  milk  ^ 

causes  the  fat  globules  to  unite  and  adhere  more     ^^  "^ 

or  less,  a  characteristic  which  is  also  noticeable 
in  pasteurized  milk.  The  flavor  and  odor  of 
boiled  milk  is  in  itself  quite  characteristic. 

Knapp  recommends  the  following  test  for 
determining  the  addition  of  water  to  milk.  10 
cc.  of  the  suspected  milk  are  run  into  a  test-tube 
and  curdled  by  adding  one  drop  of  rennet  and 
placing  the  tube  in  the  water  bath  for  about  2 
min.  at  a  temperature  of  35^-40°  C.  The 
whole  is  then  poured  upon  a  very  fine  wire  sieve 
and  the  liquid  allowed  to  drain  off  into  a  tube 
graduate,  pressing  the  curd  with  a  glass  rod  so 
as  to  remove  the  liquid  as  completely  as  possible. 
The  amount  of  liquid  remaining  in  the  curd 
is  fairly  constant  in  the  tests  and  therefore 
practically  neghgible  for  comparative  purposes,  culture'  tl'~Staphylo- 
If  the  amount  of  liquid  drained  off  exceeds  8    coccus  aureus  1  vfeek 

\  -,     -,       rx.,  .  ,        111        old.— {MacNeal.) 

CC,  water  has  been  added.  This  test  should  be 
checked  by  the  chemical  butter  fat  tests  and  also  by  the  microscop- 
ical method  for  determining  the  fat  content,  as  already  explained. 
Among  the  micro-organisms  which  cause  the  coagulation  of 
milk  and  which  are  often  found  in  sour  milk,  particularly  in  old  sour 
10 


132  BACTERIOLOGICAL  METHODS 

milk,  is  the  Streptococcus  lacticus  of  Kruse.  The  Bacillus  {lactis) 
aerogenes  which  is  very  closely  similar  to  Bacillus  colij  also  sours 
milk  and  is  likely  to  be  present  at  the  beginning  of  the  fermenta- 
tion. The  common  pus  streptococci  and  staphylococci  are  often 
found  in  milk  in  large  numbers,  traceable  to  dirt  and  filth  and  to 
diseased  udders  and  less  commonly  to  the  hands  of  the  milkers. 
The  colon  bacillus  when  present  is  traceable  to  stable  dust  and 
manure  and  to  the  unclean  hands  of  the  milkers. 

The  following  are  some  of  the  organisms  which  cause  diseases 
of  milk: 

1.  Bacillus  cyano genes — Blue  milk. 

2.  Bacillus  prodigiosus — Red  milk. 

3.  Bacillus  erythro genes — Red  milk. 

4.  Bacillus  synxanthus — Yellow  milk. 

5.  Torula  amara — Bitter  milk. 

6.  Streptococcus  hollandicus — Ropy  milk. 

Naturally  the  bacilli  normally  present  in  the  milk  which  is 
stored  for  cream  formation  are  also  present  in  the  cream  after  the 
skimming  and  cause  the  so-called  ripening  of  the  cream.  In 
order  that  the  ripening  process  may  proceed  in  a  desirable  manner, 
the  objectionable  butyric  acid  formers  must  be  excluded.  The 
butyric  acid  formers  are  more  generally  associated  with  filth,  hence, 
a  careful  compliance  with  sanitary  rules  and  regulations  in  the 
dairying  establishment  will  generally  encourage  the  invasion  and 
development  of  the  desirable  lactic  acid  organisms  to  the  exclusion 
of  the  undesirable  microbes,  though  this  is  by  no  means  always 
the  case.  Occasionally,  even  with  the  most  scrupulous  adherence 
to  sanitation,  cream  will  not  ripen  properly  and  these  occasional 
failures  have  prompted  the  more  progressive  dairymen  to  in- 
oculate the  milk  and  cream  with  pure  cultures  of  the  desirable  cream 
ripening  bacilli.  Others  use  natural  cream  starters,  that  is,  small 
quantities  of  old  cream  which  has  ripened  with  a  desirable  flavor. 

Cream  should  not  show  colon  bacilli  in  less  than  o.io  cc.  quanti- 
ties and  fresh  unripened  cream  should  not  contain  more  than  5,000,- 


TUBERCLE  BACILLI  IN  MILK  1 33 

000  bacteria  per  cc.  Ripened  cream  should  not  contain  more  than 
150,000,000  bacteria  per  cc.  and  most  of  which  bacteria  should  be 
of  the  lactic  acid  group.  Pathogenic  bacteria  which  may  be  pres- 
ent in  milk  may  also  be  present  in  the  cream.  Tubercle  bacilH, 
diphtheria  bacilH  and  typhoid  bacilli  are  the  most  likely  to  occur. 
In  the  case  of  doubtful  cream,  the  colon  bacillus  test  should  not  be 
omitted  and  in  the  case  of  suspected  contamination  with  patho- 
genic organisms,  the  cream,  as  well  as  the  milk  from  the  same 
source,  should  be  examined,  resorting  to  the 
usual  animal  inoculation  tests. 

The  tests  for  the  presence  of  tubercle  bac- 
illi in  milk,  cream^,  meats,  etc.,  comprises  the 
microscopic  examination  of  stained  (Ziehl- 
Neelsen  method  of  staining)  sediments  or  con- 
centrates as  may  be  required,  and  animal  in- 
oculations.    For  the  animal    inoculation   test,    ,  ^J,?:  42.— Tubercle 

'     bacilli     m     sputum. 

guinea-pigs  are  used.  Centrifugalize  (in  a  Stained  with  carbol- 
powerful  machine)  about  250  cc.  of  the  milk  (eneWue.— tpSje/Z) 
in  order  to  throw  down  the  tubercle  bacilli  (with 
the  other  inclusions),  and  from  this  make  the  desired  cover-slip 
preparations  and  inoculate  (in  the  region  of  the  left  knee-joint 
of  hind  leg)  the  remainder  of  the  sediment  into  three  healthy 
guinea-pigs.  Place  the  inoculated  guinea-pigs  in  individual  cages 
and  keep  them  under  observation  for  from  2  to  4  weeks.  The 
reasons  why  several  pigs  should  be  inoculated  are  as  follows. 
Some  of  the  pigs  may  be  killed  by  bacteria  other  than  the  tu- 
bercle bacilli  and  it  is  always  desirable  to  dupHcate  the  tests. 
At  the  end  of  the  second  week,  one  of  the  guinea-pigs  should  be 
dissected  and  the  glands  of  the  sublumbar  region  as  well  as  the 
glands  of  the  superficial  tissues  and  of  the  popUteal  region  exam- 
ined. If  tubercular  infection  has  taken  place,  these  glands  will 
be  found  much  enlarged  containing  foci  of  tubercle  bacilli.  The 
enlarged  glands  are  dissected  and  suitable  cover-glass  prepara- 
tions made  therefrom.     If  the  evidence  of  tubercular  infection 


134 


BACTERIOLOGICAL  METHODS 


Fig.  43. — Tubercle  ba- 
cillus slant  culture  on  glyc- 
erin-agar,  several  months 
o\d.— {suit,  after  Curtis.) 


is  not  conclusive,  the  other  two  inocu- 
lated guinea-pigs  should  be  kept  2  weeks 
longer  then  dissected  and  examined  like 
the  first.  Occasionally  there  is  abscess 
formation  at  the  point  of  inoculation  but 
this  need  not  necessarily  interfere  with  the 
tubercular  development  in  the  glands  and 
in  the  deeper  tissues. 

It  is  frequently  possible  to  isolate  the 
bacillus  of  tuberculosis  (from  sputum, 
glandular  tissues,  meat  pulp,  centrifugal- 
ized  sediments  of  milk,  cream,  etc.)  by 
special  manipulation  and  the  use  of  special 
culture  media.  The  following  method 
is  suggested.  Spread  two  or  three  drops 
of  the  material  (concentrate,  sediment, 
crushed,  suspected  tuberculous  meat  ex- 
tract, etc.)  evenly  over  the  surface  of  two 
or  three  glass  slips  and  place  the  smear 
preparations  in  the  drying  oven  at  100°  C. 
for  15  min.,  however,  not  before  the  ma- 
terial on  the  slips  is  well  dried  at  the  room 
temperature.  Tubercle  bacilli  are  quite 
resistant  to  dry  heat  and  will  withstand 
the  temperature  of  100°  C.  for  from  30 
min.  to  I  hr.  The  exposure  to  that  tem- 
perature for  15  min.  will  kill  most  of  the 
bacteria  associated  with  the  tubercle  germs 
and  will  in  fact  kill  some  of  these.  At 
the  end  of  15  min.  take  the  glass  slips 
from  the  drying  oven  and  by  means  of  a 
small  sterile  spatula  or  scalpel,  scrape  the 
dried  suspected  material  over  the  surface 


CULTURING   THE   TUBERCLE  BACILLI   OF   MILK  135 

of  the  special  medium  in  Petri  dishes.     The  medium  used  (Hesse's 
agar)  is  made  as  follows : 

Nutrose  (sodium  caseinate) 5  grams. 

Sodium  chloride 30  grams. 

Glycerin 30  grams. 

Agar 10  grams. 

Na2C02(crystalline)  solution  (28.6  per  cent.) 5  cc. 

Distilled  water 1000  cc. 

Mix  ingredients.  Heat  until  agar  is  dissolved.  Filter  through 
cotton.     Pour  into  Petri  dishes.     Sterilize  fractionally. 

After  inoculating  two  or  three  Petri  dishes  in  the  manner 
indicated,  incubate  at  37.5°  C.  in  a  moisture-saturated  atmosphere 
for  several  days.  If  tubercle  bacilli  are  present  young  colonies 
will  appear  which  may  be  identified  with  a  low  power  by  the  resem- 
blance to  broken  wavy  lines. 

Instead  of  making  the  glass-slip  smears  as  above  suggested, 
good  results  may  be  obtained  through  the  use  of  the  cotton  throat 
swabs  such  as  are  used  by  physicians  for  taking  throat  cultures  in 
diphtheria  cases.  Dip  or  roll  the  cotton  ends  of  three  swabs  in 
the  suspected  tuberculous  material,  suspend  in  air  until  perfectly 
dry  and  then  place  in  drying  over  (100°  C.)  for  15  min.,  then  rub 
the  cotton  over  the  surface  of  the  special  culture  medium  in  Petri 
dishes.  Make  some  six  or  seven  parallel  streaks  over  the  surface  of 
the  medium.  Incubate  and  examine  as  before.  Should  the  glass- 
slip  or  cotton-swab  preparations  be  placed  in  the  drying  oven 
before  air  drying  them,  all  or  nearly  all  of  the  tubercle  bacilli  would 
be  killed  in  the  drying  oven. 

Several  investigators  have  recommended  a  direct  method  of 
examination  for  ascertaining  the  presence  of  tubercle  bacilli  in 
milk,  and  in  other  materials,  through  the  use  of  agents  which  will 
completely  dissolve  all  bacterial  bodies  excepting  the  acid-fast 
group  of  organisms  to  which  the  tubercle  bacillus  belongs.  For 
this  purpose  antiformin  (really  a  mixture  of  chlorinated  sodium 
hypochlorite  and  Labarraque's  solution)  has  been  highly  recom- 


136  BACTERIOLOGICAL  METHODS 

mended.  This  proprietary  article  is  a  strongly  alkaline  solution  of 
sodium  hypochlorite.  In  each  cc.  it  contains  approximately  5.68 
grams  of  sodium  hypochlorite,  sodium  hydroxide  7.8  grams  and 
sodium  carbonate  0.32  grams.  The  available  chlorine  amounts  to 
about  5.68  grams.     It  dissolves  all  organic  matter,  such  as  that  con- 


FiG.  44. — Bacillus  tuberculosis  in  the  sputum  of  a  consumptive;  stained  by  Ziehl 
method  (X  2100). — (After  Kossel.) 

tained  in  sputum  and  feces,  excepting  the  tubercle  bacilli.     It  is  in 
itself  an  active  antiseptic  having  a  phenol  coefficient  of  3. 

For  bacteriological  work,  a  50  per  cent,  solution  of  the  anti- 
formin  will  be  found  satisfactory.  Mix  equal  parts  of  the  anti- 
formin  solution  (50  per  cent.)  and  milk  or  sputum  or  other  mate- 


ANTIFORMIN  TUBERCLE  CULTURES  137 

Irial  supposed  to  contain  the  tubercle  bacilli,  in  a  suitable  glass 
container  and  bring  to  a  boil  over  the  Bunsen  burner.  When  the 
material  is  cool,  add  1.5  cc.  of  a  mixture  of  chloroform  and  alcohol 
(chloroform  one  part  and  alcohol  nine  parts)  to  each  lo  cc.  of  the 
material  and  shake  vigorously.  The  tubercle  bacilli  absorb  some 
of  the  chloroform  and  become  heavier  than  the  rest  of  the  organic 
matter.  Next  centrifugalize  at  a  high  speed  for  15  min.  which 
separates  the  material  into  three  layers;  the  antiformin  at  the  top, 
the  sediment  in  the  middle,  and  the  chloroform  with  the  tubercle 
bacilli  at  the  bottom.  Pipette  off  the  layer  of  chloroform  and  ex- 
amine for  tubercle  bacilli  by  resorting  to  the  usual  staining  methods. 
The  smear  preparations  can  be  made  to  stick  to  the  cover  or  slide 
by  mixing  with  serum  or  egg  albumen  solution.  This  method  may 
also  be  tried  in  the  examination  of  creams,  cheese,  buttermilk  and 
butter.  The  strength  of  the  antiformin  solution  should  be  graded 
according  to  the  amount  or  percentage  of  organic  matter  to  be  dis- 
solved, taking  the  strength  required  for  sputum  work  as  the  high- 
est. For  milk  work  the  15  per  cent,  solution  will  be  satisfactory. 
For  cheese  a  50  per  cent,  solution  should  be  used,  likewise  for 
feces. 

Stitt  recommends  the  following  antiformin  method  for  cultur- 
ing  the  tubercle  bacilli.  Mix  20  cc.  of  sputum,  65  cc.  of  sterile 
water  and  15  cc.  of  antiformin.  Stir  with  a  glass  rod.  After  a 
period  ranging  from  30  min.  to  2  hr.,  the  mixture  should  be 
homogeneous.  Centrifugalize  for  15  min.  or  longer,  decant,  and 
wash  the  sediment  twice  in  sterile  normal  salt  solution  and  smear 
out  the  well-washed  sediment  over  serum  or  glycerin  egg  albumen 
or  nutrose  slants.  It  must  be  remembered  that  the  tubercle  bacillus 
will  not  grow  in  sunlight  and  that  the  colonies  form  on  the  surface 
of  the  culture  media  only. 

Stitt  also  states  that  it  is  not  wise  to  use  the  antiformin  in 
solutions  stronger  than  is  necessary  to  dissolve  the  organic  matter 
and  bacteria  other  than  the  tubercle  bacillus.  For  example  for 
sputum,  it  is  suggested  that  20  or  25  per  cent,  of  antiformin  be 


138  BACTERIOLOGICAL  METHODS 

used.  If  stronger  solutions  are  used,  many  of  the  tubercle  bacilli 
are  also  disintegrated  or  considerably  changed  in  form  and  in  the 
behavior  with  the  acid-fast  stains. 

In  addition  to  the  routine  examination  of  ice  creams  for  the 
presence  of  fillers  and  ingredients  which  do  not  properly  belong 
to  ice  creams,  the  food  bacteriologist  will  have  occasion  to  make 
bacteriological  and  toxicological  tests.  According  to  Vaughan, 
the  toxic  changes  in  ice  cream  are  due  to  the  presence  of  a  poison 
designated  tyrotoxicon,  presumably  identical  with  the  toxin  occa- 
sionally found  in  milk  and  cheese.  Ice-cream  poisoning  depends 
upon  the  development  of  the  toxin-forming  bacteria  in  the  milk 
and  cream  before  it  is  frozen.  It  is  not  at  all  likely  that  ice  cream 
made  from  clean  wholesome  cream  and  milk  will  contain  toxins, 
provided  it  is  kept  well  frozen  and  is  not  stored  too  long.  There 
is  good  evidence  that  slightly  infected  ice  cream  which  is  kept  for 
several  days  and  longer,  may  show  sufficient  toxic  bacterial  de- 
velopment to  produce  symptoms  of  poisoning.  The  virulency  of 
the  toxins  produced  by  the  bacteria  appears  to  increase  with  the 
lowering  of  the  temperature. 

The  danger  from  ice  cream  is  directly  proportional  to  the  un- 
sanitary conditions  of  the  milk  and  cream  used  and  ice-cream 
poisoning  is  far  more  likely  to  manifest  itself  during  the  hot  summer 
weather.  All  suspicious  ice  creams  should  be  examined  bacteri- 
ally,  making  numerical  plate  cultures  and  also  the  presumptive  co- 
lon bacillus  test  and  tests  for  streptococci  and  staphylococci.  The 
toxicological  test  as  recommended  for  meats,  is,  however,  far  more 
important  and  should  not  be  omitted.  Ice  cream  should  not  con- 
tain more  than  1,000,000  bacteria  per  cc.  and  should  not  develop 
colon  bacilli  in  less  than  o.io  cc.  quantities  by  the  standard  pre- 
sumptive colon  bacillus  test. 

Of  the  more  common  ice-cream  fillers  we  may  mention  starch 
and  tragacanth.  Vegetable  mucilages  other  than  tragacanth  may 
be  suspected.  Gelatin  is  also  used.  Eggs  are  frequently  added. 
A  filler  to  which  a  small  amount  of  rennet  had  been  added  has 


BUTTER  AND   CHEESE  139 

been  extensively  advertised  as  an  ice-cream  producer  which  did 
not  require  the  use  of  cream  or  of  ice. 

Occasionally  it  may  become  necessary  to  examine  sour  milk 
and  buttermilk  for  the  presence  of  toxins  and  objectionable  bac- 
teria and  other  undesirable  organisms.  Because  of  the  careless 
and  more  or  less  promiscuous  handling  of  buttermilk  before  it 
reaches  the  consumer,  it  is  especially  liable  to  the  invasion  of 
foreign  organisms.  The  routine  examination  of  buttermilk  is 
largely  limited  to  a  direct  microscopical  inspection.  Mold  spores 
and  yeast  cells  should  be  sparingly  present  and  the  predominating 
bacilli  should  be  small^  (lactic  acid  formers)  and  of  irregular  and 
rather  indefinite  outline.  Mold  and  cocci  should  be  very  sparingly 
present. 

To  examine  butter  for  the  presence  of  bacteria  (direct  micro- 
scopical method)  and  other  contaminations,  place  i  gram  of  the 
butter  in  10  cc.  of  ether  and  shake  until  all  of  the  butter  fat  is 
dissolved.  Pour  the  solution  into  the  special  centrifugal  tube 
and  centrifugalize  for  5  min.  Wash  the  contents  of  the  i  cc. 
end  tube  into  10  cc.  of  ether  and  again  shake  and  centrifugaHze. 
Pour  off  the  ether  and  add  2  cc.  of  a  2  per  cent,  sodic  hydrate 
solution  and  shake  until  the  casein  is  dissolved.  The  sodic  hydrate 
solution  emulsifies  the  small  amount  of  fat  present.  Examine 
the  emulsion  for  bacteria,  counting  the  bacteria  and  body  cells 
by  means  of  the  hemacytometer. 

Butter  and  cheese  made  from  the  milk  of  animals  suffering 
from  foot-and-mouth  disease  have  transmitted  this  disease  to 
humans.  The  bovine  type  of  tuberculosis  has  resulted  from  the 
consumption  of  milk,  cream  and  butter.  Tubercle  bacilli  have 
been  found  in  the  more  quickly  ripened  cheeses.  Tubercle  bacilli 
do,  however,  not  survive  long  in  soured  cream  or  milk,  perhaps 
not  over  2  or  3  days. 

The  following  are  some  of  the  more  important  organisms  con- 
cerned in  the  ripening  of  cheese. 

^  The  Bacillus  bulgarius  is  comparatively  large  (i  X  6/*). 


I40  BACTERIOLOGICAL  METHODS 

1.  Lactic  acid  bacteria. — These  are  the  chief  agents  concerned  in  the  ripening  of 
Cheddar,  American  and  Edam  cheese.  Pure  cultures  of  the  Bacillus  acidi  lactici  are 
often  used  as  a  starter.  In  the  manufacture  of  the  Edam  cheese,  slimy  whey  is 
used  as  a  starter  {Streptococcus  hollandicus) . 

2.  Penicillium  glaucum  the  common  green  mold  is  the  principle  organism  con- 
cerned in  the  ripening  of  Roquefort,  Gorganzola  and  Brie  cheeses.  In  some  coun- 
tries the  green  mold  is  scraped  from  molded  bread  and  added  to  the  curd. 

3.  A  great  variety  of  other  bacteria,  yeasts  and  mold  are  concerned  in  the  devel- 
opment of  the  more  specific  flavors  and  aromas.  Further  investigation  is  necessary 
to  ascertain  the  special  function  performed  by  each  and  the  mutualistic  relationship 
that  may  exist  between  them. 

4.  Gas  generating  bacteria  are  concerned  in  the  formation  of  holes  in  the  interior 
of  the  ripening  cheese.  These  gas  formers  also  modify  the  aroma  or  flavor  of  the 
cheese  and  in  some  instances  constitute  the  chief  ripening  agents. 

Spoiling  of  cheese  is  not  uncommon,  due  to  the  invasion  of  a 
variety  of  undesirable  organisms.  The  cheese  ''hopper"  or 
"skipper"  found  in  and  upon  old  and  overripened  cheese  and  in 
cheeses  which  have  not  been  properly  screened,  is  the  larva  of  the 
black  two-winged  fly  Piophila  casei.  The  insect  deposits  its 
eggs  in  the  surface  cracks  and  crevices  of  the  cheese  upon  which  the 
developing  larva  feeds.  The  name  skipper  or  hopper  is  derived 
from  the  fact  that  the  larvae  are  capable  of  projecting  themselves 
some  distance  by  coiling  and  suddenly  uncoiling. 

This  fly  is  a  common  pest  in  the  dairying  establishments.  A 
less  common  but  even  more  annoying  pest  is  the  larva  of  the 
''bacon  beetle."  Cheeses  which  are  comparatively  hard  and 
smooth  externally  are  not  so  likely  to  be  infested  by  the  skipper 
or  bacon  beetle  larva  as  are  the  cheeses  which  are  rough  externally. 
It  is  customary  to  wipe  the  cheese  in  order  to  remove  the  para- 
sites. If  the  cheeses  which  are  stored  for  ripening  are  properly 
screened,  the  fly  and  beetle  cannot  get  access  to  them  to  deposit 
the  eggs.  A  small  mite  (Trioglyphis  siro)  also  occurs  on  cheese 
upon  which  it  feeds. 

Inadequately  screened  cheeses  also  permit  flies  and  other  pests 
to  deposit  possible  infections,  thus  typhoid  contamination  and 
also  pus  streptococci  and  staphylococci  may  be  found  upon  this 
food  substance. 


DISEASES   OF   CHEESE 


141 


Bitter  cheese  is  due  to  a  variety  of  bacteria,  as  Tyrothrix 
geniculatus  (the  bitter  soft  cheese  bacillus),  Micrococcus  casei  amari 
(bitter  cheese  coccus),  Weigmann's  bitter  milk  bacillus,  Conn's 
bitter  milk  coccus,  and  others.  Red  coloration  of  cheese  may  be 
caused  by  yeasts  (Saccharomyces  ruber)  or  by  cocci.     Black  cheese 


Fig.  45. — Oidium  lactis.  a,  b,  Dichotomous  branching  of  growing  hyphae;  c,  d,  g, 
simple  chains  of  oidia  breaking  through  substratum  at  dotted  line  x-y,  dotted  por- 
tions submerged;  e,  f,  chains  of  oidia  from  a  branching  outgrowth  of  a  submerged 
cell;  h,  branching  chain  of  oidia;  k,  I,  m,  n,  0,  p,  s,  types  of  germination  of  oidia  under 
varying  conditions;  /,  diagram  of  a  portion  of  a  colony  showing  habit  of  Oidium 
lactis  as  seen  in  culture  media. — {From  Bull.  82,  Bur.  Animal  Industry,  U.  S.  Dept. 
Agr.) 


may  be  due  to  the  presence  of  iron  in  milk,  perhaps  traceable  to  the 
action  of  slightly  soured  milk  in  rusty  buckets.  Some  yeasts  and 
molds  may  produce  dark  to  black  decomposition  changes.  Blue 
cheese  is  the  result  of  the  action  of  a  bacillus.  Putrid  cheese  is  the 
result  of  the  invasion  of  saprophytic  bacteria  and  other  micro- 


142 


BACTERIOLOGICAL  METHODS 


organisms.  Cheese  poisoning  is  not  uncommon,  due  to  the  pres- 
ence of  bacteria  which  give  rise  to  toxins  (tyro-toxicon) .  In  a 
general  way  it  may  be  stated  that  cheese  diseases  are  due  to 
filthy  and  unsanitary  conditions  in  the  dairying  establishment  re- 


FiG.  46. — Penicillium  glaucum  showing  the  characteristic  spore  formation.  This 
fungus  is  a  true  saprophyte,  the  common  green  mold,  occurring  on  a  great  variety 
of  organic  substances. 

suiting  in  infected  milk,  or  to  filthy  and  unsanitary  conditions  in 
the  cheese  factory,  or  the  infection  may  be  traceable  to  the  im- 
proper and  careless  storing  and  handling  of  the  cheese.  Ripened 
cheese  being  in  itself  a  decomposition  product  resulting  from  the 
invasion  of  certain  desirable   micro-organisms   usually   entering 


CANNED   AND    CONDENSED   MILK 


143 


from  the  air,  it  is  but  reasonable  to  expect  irregularities  in  the  final 
result  unless  the  invasion  of  undesirable  micro-organisms,  which 
are  also  present  in  the  air,  is  carefully  guarded  against. 

Condensed  milk  is  prepared  by  concentrating  full  or  skimmed 
milk.  It  may  be  sweetened  by  adding  cane  sugar  (40  per  cent.). 
While  condensed  milk  contains  relatively  fewer  bacteria  than  does 
ordinary  milk,  due  to  the  process  of  manufacture,  yet  none  is 
entirely  sterile.  The  number  of  bacteria  usually  present  ranges 
from  about  500  or  even  less  to  as  high  as 
250,000  per  cc.  Colon  bacilli,  dysentery 
bacilH  and  streptococci  are  generally 
absent.  Tubercle  bacilli  have  been  found. 
The  method  for  examining  condensed  milk 
is  much  as  for  ordinary  milk,  with  suitable 
modifications  in  making  the  dilutions. 

Canned  condensed  milk  occasionally 
spoils,  due  to  the  development  of  bacteria 
and  yeast  organisms.  Yeast  organisms  are 
not  likely  to  appear  unless  the  milk  is 
sweetened  with  sugar.  Spoiling  may  be- 
come apparent  through  the  "swelling"  of 
the  can.  Organoleptic  testing  is  occasion- 
ally a  guide  to  the  condition  or  quaHty  of 
the  milk.  A  numerical  bacterial  limit 
should  be  adopted  for  condensed  milk.  If 
more  than  1,000,000  bacteria  per  cc.  are 
present  it  is  not  suitable  for  human  consumption.  Tubercle  bacilli 
should  be  absent.  According  to  the  limited  reports  on  the  subject 
we  may  assume  that  the  process  of  condensing  the  milk  kills  all 
pathogenic  bacteria  which  may  be  present,  including  even  the  more 
resistant  tubercle  bacilli.  The  contaminating  bacteria  may  pro- 
duce toxins  and  in  marked  bacterial  invasion  it  would  be  well  to 
make  inoculation  tests  with  white  mice  or  guinea-pigs,  as  for  toxins 
in  meat  and  in  ice  cream.     An  examination  of  the  centrifugalized 


Fig.  47. — Penicillium 
of  Camembert  and  Roque- 
fart  cheese.  This  mold 
grows  at  a  very  low  tem- 
perature. It  is  closely 
similar  to,  if  not  identical, 
with  P.  glaucum. — {Jor- 
dan after  Thorn.) 


144  BACTERIOLOGICAL  METHODS 

sediment  must  not  be  omitted  as  this  will  convey  information  re- 
garding the  sanitary  conditions  of  the  factory  as  well  as  of  the 
dairying  establishments  which  supplied  the  milk  to  the  factory. 

Dried  or  powdered  milk  is  prepared  by  spraying  milk  (usually 
skimmed)  into  a  partial  vacuum  or  by  spraying  it  on  a  revolving 
drum  or  on  a  moving  belt  in  a  partial  vacuum.  The  dried  material 
is  then  placed  in  suitable  containers.  The  dried  milk  contains 
all  of  the  ingredients  of  the  milk  excepting  the  water,  the  lysins 
and  certain  enzymes.  The  fat  globules  are  altered  physically 
but  not  chemically.  Mixing  dried  milk  with  the  required  amounts 
of  water  makes  a  liquid  resembling  ordinary  milk.  The  micro- 
scopical and  bacteriological  examination  of  dried  milk  is  as  for 
condensed  milk.  Like  the  condensed  milk  it  is  quite  free  from 
disease  germs  of  all  kinds  but  bacterial  invasion  is  not  excluded 
from  material  which  has  been  carelessly  prepared  or  canned. 
Toxins  and  ptomaines  should  be  absent.  The  absence  of  moisture 
in  powdered  milk  prevents  the  ready  growth  of  micro-organisms 
and  it  may  be  kept  in  good  condition  for  a  period  of  5  or  6  months 
and  even  longer,  in  dry  sterile  containers  stored  in  a  cool  dry 
place. 

Attempts  have  been  made  to  commercialize  frozen  milk  but 
so  far  without  success.  It  is  rather  difficult  to  handle  frozen 
milk  and  the  article  furthermore  loses  the  milk  flavor  on  thawing. 

16.  The  Bacteriological  Examination  of  Shellfish 

The  term  shellfish  includes  oysters,  mussels  and  clams.  Only 
those  species  and  varieties  which  serve  as  food  for  man  are  of 
interest  to  the  food  bacteriologist.  Since  it  has  been  conclusively 
proven  that  shellfish,  oysters  in  particular,  have  been  responsible 
for  typhoid  epidemics,  much  attention  has  been  given  to  the 
bacteriology  of  this  class  of  food.  In  tracing  such  epidemics  it  was 
discovered  that  the  causative  oysters  had  been  floated  or  grown  in 
heavily  polluted  waters.     In  several  instances  contamination  of 


SHELLFISH  1 45 

the  water  supply  washing  the  oyster  beds,  was  traceable  to  the 
discharges  from  typhoid  fever  patients. 

All  shellfish  are  easily  adaptable  to  filthy  habits  and  surround- 
ings. They  appear  to  thrive  in  proportion  to  the  amount  of  or- 
ganic contamination  of  the  water  supply  constituting  the  food 
beds.  It  must,  however,  not  be  supposed  that  sewage  and  other 
highly  objectionable  (to  man)  contamination  is  normal  to  the  life 
of  the  shellfish.  We  know  that  the  domestic  hog  is  fond  of  the 
highly  contaminated  refuse  materials  from  the  kitchen  known  as 
swill  but  we  also  know  that  hogs  thrive  better  on  sanitary  food. 
Thus  the  filth  feeding  oyster  grows  equally  well,  if  not  better,  in 
clean  sea  water,  that  is,  water  free  from  sewage  contamination  and 
decayed  animal  matter. 

The  danger  from  shellfish  (to  man)  is  due  to  the  fact  that  these 
animals  are  often  from  highly  contaminated  water  supplies  and 
that  they  are  generally  eaten  raw  or  only  partially  cooked.  The 
possible  diseases  traceable  to  the  eating  of  shellfish  are  Asiatic 
cholera  (in  countries  where  this  disease  prevails),  typhoid  fever 
and  a  variety  of  less  severe  intestinal  diseases  such  as  dysentery, 
colitis  and  intestinal  ulcerations.  The  work  of  the  food  bacteri- 
ologist is,  however,  not  the  finding  of  the  specific  germs  causing  an 
epidemic,  but  rather  an  endeavor  to  ascertain  the  danger  point  in 
the  quality  of  the  food  as  represented  by  the  positive  colon  bacillus 
tests.  The  prime  object  of  the  pure  food  laws  is  the  maintenance 
of  health  rather  than  finding  the  cause  of  disease.  This  most 
important  fact  is  sometimes  not  understood  as  is  clearly  indicated 
by  a  supreme  court  decision  permitting  the  bleaching  of  flour. 
It  is  the  intent  of  the  pure  food  law  to  clearly  mark  the  danger 
points  in  our  food  supplies  so  that  the  consumer  may  maintain 
his  physical  well-being  through  the  avoidance  of  such  dangers. 
He  who  advises  against  the  heeding  of  the  proper  and  timely  warn- 
ings set  up  by  those  entrusted  with  this  duty,  either  through 
ignorance  or  indifference,  is  a  menace  to  the  public  welfare.  The 
danger  sign,  "avoid  bleached  flour''  and  not  the  actual  physical 


146  BACTERIOLOGICAL  METHODS 

disturbances  which  results  from  the  eating  of  such  flour,  is  the 
proper  warning.  The  presence  of  a  limited  number  of  colon  bacilli 
in  foods  and  drinks  is  the  danger  mark  and  not  the  actual  occur- 
rence of  cholera,  of  typhoid,  of  dysentery,  due  to  the  eating  of 
more  highly  contaminated  foods.  The  danger  signal  must  be 
within  the  zone  of  safety  and  not  beyond  it. 

The  methods  for  the  bacteriological  examination  of  shellfish 
are  but  modifications  of  the  methods  used  in  the  examination  of 
water  supplies.  As  in  the  case  of  drinking  water,  the  chief  index 
to  the  pollution  of  shellfish  is  the  colon  bacillus  test.  The  examina- 
tion of  the  water  source  above  the  oyster  beds  very  frequently 
gives  inferential  information  as  to  the  possibility  of  the  contami- 
nation of  the  shellfish  which  obtain  their  food  supply  from  beds 
flooded  by  such  waters.  The  following  is  the  method  for  the 
bacteriological  examination  of  shellfish  adopted  by  the  American 
Health  Association  at  the  191 2  meeting. 

1.  Selection  of  Sample.^ — Twelve  oysters  of  average  size  of  the 
lot  to  be  examined,  having  deep  bowls,  short  lips  and  shell  tightly 
closed,  are  picked  out  by  hand  or  by  means  of  a  sterilized  long- 
handled  spoon  and  prepared  for  immediate  transportation  to  the 
laboratory. 

2.  Making  a  Record  of  the  Sample.^ — This  record  should  cover 
the  following  points.  The  exact  location  of  the  bed  from  which  the 
sample  was  taken.  The  depth  of  the  water  at  the  time  the  oysters 
were  gathered.  Weather  conditions,  direction  and  velocity  of 
wind,  state  of  tide,  day  and  hour  when  the  stock  was  taken  from 
the  water,  the  conditions  under  which  the  stock  had  been  kept 
since  removal  from  the  water  and  up  to  the  time  when  the  sample 
was  taken,  presence  of  abnormal  odors,  temperature  of  stock,  and 
the  day  and  hour  of  taking  the  sample. 

3.  Transportation  of  the  Sample. — The  sample  oysters  are  to 
be  packed  in  a  suitable  metal  or  pasteboard  container  of  the  size 
and  shape  convenient  for  shipping.  The  important  points  to  bear 
in  mind  are:  the  prevention  of  the  mixing  of  the  oyster  liquors  of 


SHELLFISH 


147 


the  different  samples  and  avoiding  the  mixing  of  the  oysters  with 
the  ice  water  of  the  packing  ice.  The  samples  must  in  all  cases  be 
placed  on  ice  or  packed  in  ice  if  they  cannot  be  examined  inside  of 
36  hr.  or  if  the  outside  temperature  is  above  50°  F.  It  is/however, 
not  necessary  to  place  the  oysters  in  absolutely  tight  containers 
provided  the  above  conditions  are  maintained. 

4.  Laboratory  Procedure. — Record  the  date  of  receiving  the 
sample,  condition  of  seals,  of  the  sample  oysters  and  the  tem- 
perature of  the  interior  of  the  container  at  the  time  of  opening. 
The  bacteriological  examination  should  in  all  cases  be  started  as 
soon  as  possible  after  the  receipt  of  the  sample. 

Before  beginning  operations  the  hands  must  be  thoroughly 
scrubbed  and  all  vessels  to  be  used  must  be  sterilized.  The  shell 
of  the  oyster  may  be  opened  by  means  of  a  sterilized  oyster  knife 
or  by  drilling  a  hole  through  the  shell  near  the  hinge.  The  drill 
must  be  sterilized  and  the  area  of  the  shell  to  be  operated  upon  must 
be  cleaned,  flamed  before  drilling  and  flamed  at  least  once  more 
during   the    drilling   process. 

The  simplest  and  quickest  method  for  opening  the  oyster  shells 
is  that  employed  by  Stiles  of  the  Bureau  of  Chemistry.  By  means 
of  a  pair  of  sterilized  wire  nippers  crush  and  break  off  enough  of 
the  two  valves  so  as  to  make  the  use  of  the  oyster  knife  easy. 

Before  opening  the  oysters  see  that  they  are  thoroughly 
scrubbed  and  then  rinsed  in  boiled  (sterile)  water,  and  each 
oyster  is  wiped  quite  dry  and  flamed  before  it  is  opened. 

5.  Bacterial  Counts. — Bacterial  counts  are  made  of  the  compos- 
ite sample  of  each  lot  obtained  by  mixing  the  shell  Hquor  of  five 
oysters.  Agar  shall  be  used  for  the  culture  medium  and  in  general 
the  procedure  shall  be  in  accordance  with  the  method  recommended 
for  the  examination  of  water.  The  water  used  for  making  the  di- 
lutions shall  contain  i  per  cent,  of  sodium  chloride,  in  order  to 
approximate  the  natural  saHnity  of  the  oyster  liquor.  The  agar 
plate  cultures  shall  be  incubated  at  20°  C.  for  3  days  and  the  col- 
onies counted  in  the  usual  manner. 


148  BACTERIOLOGICAL  METHODS 

6.  Determining  Bacteria  of  the  Colon  Bacillus  Group. — Meas- 
ured quantities  of  the  shell  liquor  of  each  of  five  oysters  selected 
from  the  dozen  shall  be  placed  in  fermentation  tubes  containing 
lactose-peptone-bile.  The  measured  quantities  shall  be  i  cc, 
o.io  cc,  and  o.oi  cc,  or  such  other  quantities  or  corresponding 
dilutions  as  may  be  desired.  The  fermentation  tube  inoculations 
thus  prepared  shall  be  incubated  for  3  days  at  a  temperature  of  37° 
C,  and  the  presence  of  gas  noted  daily.  From  10  to  85  per  cent, 
of  gas  during  this  period  shall  be  considered  a  positive  test  indi- 
cating a  presumption  of  the  presence  of  at  least  one  bacterium  of 
the  colon  bacillus  group  in  the  quantity  of  the  water  used  in  the 
test.  But  no  final  colon  bacillus  rating  shall  be  made  unless  con- 
firmatory tests  for  the  presence  of  organisms  of  the  colon  bacillus 
group  shall  have  been  obtained  from  the  tube  of  highest  or  next 
highest  dilution  from  each  oyster  showing  the  presence  of  gas. 
These  confirmatory  tests  shall  be  begun  immediately  upon  noting 
the  formation  of  gas  and  shall  be  carried  out  in  conformity  with 
the  procedure  recommended  by  the  Committee  on  Standard 
Methods  of  Water  Analysis. 

7.  Statement  of  Results. — The  results  of  the  bacterial  counts 
shall  be  expressed  as  the  number  of  bacteria  per  cc.  The  results 
of  the  colon  bacillus  test  shall  be  expressed  either  in  the  form  of  an 
arbitrary  numerical  system  or  in  estimated  number  of  colon  bacilH 
per  cc.  of  the  sample. 

It  is  suggested  that  the  arbitrary  numerical  method  proposed 
by  the  American  Health  Association  be  given  the  preference.  The 
following  are  the  rating  valuations  according  to  this  method. 

Colon  bacillus  in  i  .00  cc.  but  not  in  o.  10  cc,  a  value  of  i 
Colon  bacillus  in  o.  10  cc.  but  not  in  o.oi    cc,  a  value  of  10 
Colon  bacillus  in  o.oi  cc  but  not  in  o.ooi  cc,  a  value  of  100,  etc. 

The  sum  of  these  values  for  five  oysters  gives  the  total  value  of 
the  sample  examined  and  this  figure  indicates  the  rating  for 
Bacillus  coli.     According  to  this  system  the  highest  (best)  rating 


THE   RATING   OF   SHELLFISH 


149 


is  indicated  by  o  and  the  lowest  (worst)  by  500,  represented  in 
tabular  form  by  the  following  possible  results  of  two  analyses: 


Example  A 


Oysters 


Numerical 
Value 


Total  rating  for  B.  coli  = 


Example  B 


Oysters 

1. 00  cc. 

O.IOCC, 

o.oi  cc. 

Numerical 
Value 

I 

+ 

+ 

+ 

100 

2 

+ 

+ 

+ 

100 

3 

+ 

+ 

+ 

100 

4 

+ 

+ 

+ 

100 

S 

+ 

+ 

+ 

100 

Total  rating  for  B.  coli   = 

Soo 

The  (+)  mark  means  that  gas  formation  in  the  lactose  bile  tubes  took  place, 
indicating  contamination  with  the  colon  bacillus. 

The  (o)  mark  indicates  that  no  gas  formation  took  place  in  the  lactose  bile  tubes. 

The  results  above  indicated  are,  however,  not  generally  ob- 
tained in  practice.  The  important  question  is  at  what  rating 
shall  the  shellfish  be  pronounced  unfit  for  human  use,  or  rather 
what  rating  shall  be  the  danger  signal  as  to  the  quality  of  this  food? 
There  seems  to  be  no  uniformity  of  opinion  as  regards  this  point. 
Thus  far,  the  Bureau  of  Chemistry  has  barred  oysters  from  inter- 
state shipment  which  gave  three  positive  tests  out  of  five  in  o.io 
cc.  quantities  of  oyster  liquor,  which  standard  is  also  adopted  by 


I50 


BACTERIOLOGICAL  METHODS 


the  Rhode  Island  Shellfish  Commission.     This  standard  may  be 
graphically  represented  as  follows  (Example  C) : 


Example  C 


Oysters 

I.OOCC. 

O.IO  cc. 

o.oi  cc. 

Numerical 
Value 

I 

+ 

+ 

o 

lO 

2 

+ 

+ 

o 

lo 

3 

+ 

+ 

o 

lO 

4 

+ 

o 

o 

I 

5 

+ 

o 

o 

I 

1 
Total  rating  for  B.  coli  = 

32 

It  sometimes  happens  in  laboratory  practice  that  the  smaller 
quantities  of  shell  water  from  a  number  of  oysters  show  positive 
results,  whereas  larger  amounts  of  liquor  from  an  equal  number  of 
oysters  show  negative  results.  In  such  cases  it  is  customary  to 
give  the  next  lower  numerical  value  to  the  positive  results  in  the 
high  dilutions,  and  such  positive  results  shall  be  considered  as 
being  transferred  to  a  lower  dilution  giving  negative  results  in 
another  oyster.  This  recession  of  assigned  values  shall,  however, 
not  be  carried  beyond  the  point  where  the  number  of  such  reces- 
sions is  greater  than  the  number  of  instances  where  other  oysters 
in  the  series  of  five  failed  to  give  positive  results.  This  may  be 
illustrated  as  follows  (Examples  D  and  E) : 

Example  D 


Oysters 

1. 00  CO. 

O.IO  cc. 

O.OI  cc. 

Numerical 

Value 

I 

+ 

+ 

o 

lO 

2 

+ 

+ 

o 

lO 

3 

+ 

+ 

o 

ID 

4 

+ 

o 

o 

lo  (not  i) 

5 

+ 

+ 

+ 

lo  (not  loo) 

1 
Total  rating  for  B.  coli  = 

SO 

THE   RATING   OF   SHELLFISH 
Example  E 


151 


Oysters 

I. 00  cc. 

o.io  cc. 

o.oi  cc. 

Numerical 
Value 

I 

+ 

+ 

+ 

10  (not  100) 

2 

+ 

+ 

+ 

10  (not  100) 

3 

+ 

0 

0 

I 

4 

0 

0 

0 

I  (not  0) 

5 

0 

0 

0 

I  (not  0) 

i 
Total  rating  for  B.  coli   = 

23 

The  bacteriological  examination  of  oysters  from  opened  or 
shucked  stock  very  naturally  must  be  somewhat  modified  from  the 
method  as  outlined  for  oysters  in  the  shell.  The  stock  in  the  con- 
tainer from  which  the  sample  is  to  be  taken  must  be  thoroughly 
mixed.  The  containers  (wide-mouthed  glass  jars)  must  be  steril- 
ized and  should  have  a  capacity  of  i  quart.  By  means  of  a  suit- 
able sterilized  ladle  (may  be  flamed  with  alcohol  on  the  spot), 
half  fill  the  containers  with  the  oysters  and  seal  containers  in  such 
manner  as  to  exclude  all  outside  contamination.  Unless  the  exami- 
nation can  be  made  within  3  hr.  after  taking  the  sample,  said 
sample  must  be  placed  on  ice.  It  is  very  desirable  to  make  the 
bacteriological  examination  shortly  after  the  sample  is  taken. 
The  laboratory  technique  is  much  as  for  oysters  in  the  shell,  though 
it  must  be  borne  in  mind  that  dilutions  higher  than  o.oi  cc.  are 
usually  required.  The  results  of  the  bacteriological  examination 
of  the  opened  or  shucked  stock  shall  be  expressed  in  the  same  way 
as  that  specified  for  oysters  in  the  shell,  except  that  in  the  calcula- 
tion for  B.  coli  rating  the  values  for  the  results  of  the  positive 
fermentation  tests,  after  confirmation,  shall  be  recorded  for  each 
of  the  inoculations  of  each  and  every  dilution.  All  tests  are  to 
be  made  in  triplicate,  that  is,  three  fermentation  tubes  are  to  be 
inoculated  for  each  dilution  used. 

Clams,  mussels  and  other  shellfish  are  to  be  examined  in  the 
same  manner  as  oysters,  in  so  far  as  this  is  possible.     In  opening 


152  BACTERIOLOGICAL   METHODS 

soft-shelled  clams  it  will  be  found  that  if  two  incisions  are  made 
through  the  mantle  the  shell  water  may  be  poured  out  without 
opening  the  shell.  It  is  stated  that  hard-shell  clams  may  be 
opened  by  striking  the  shell  over  the  dorsal  muscle  with  a  hammer. 
An  opening  is  formed  which  will  permit  the  insertion  of  a  knife 
with  which  to  cut  the  muscle.  In  case  any  one  shellfish  does  not 
contain  enough  shell  water  to  make  a  test,  the  water  from  several 
individuals  may  be  mixed. 

The  examination  of  shellfish  for  sewage  pollution  is  of  the  utmost 
importance,  as  dangerously  contaminated  oysters  are  very  com- 
mon. In  fact  it  would  be  advisable  to  discontinue  the  oyster  as 
an  article  of  diet.  At  its  very  best  it  is  a  filthy  article.  It  is 
unquestionably  a  dangerous  article  of  food,  in  this  regard  compar- 
able to  the  mushrooms  in  the  vegetable  kingdom.  However,  there 
is  not  the  least  likelihood  that  the  oyster  will  be  left  from  our 
dining  tables  as  long  as  there  are  any  available.  It  is  therefore 
most  desirable  that  the  supervising  of  this  food  on  the  part  of 
those  who  are  entrusted  with  the  safeguarding  of  the  health  of  the 
people  should  be  carefully  and  consistently  done. 

Some  authorities  (EngHsh)  recommend  that  the  liquor  and 
oysters  be  mixed,  the  latter  finely  chopped,  for  the  purpose  of 
making  the  colon  bacillus  test.  There  appears  to  be  no  gain  from 
this  procedure  and  the  method  cannot  be  recommended. 

17.  The  Bacteriological  and    Toxicological  Examination  of  Meat 
and  Meat  Products 

Remarkable  as  it  may  seem,  food  bacteriologists  have  given  but 
little  attention  to  the  examination  of  meats  and  meat  products, 
despite  the  fact  that  fatal  poisoning  from  eating  infected  meats  is 
very  common.  Intoxications  ranging  from  mild  to  very  severe, 
resulting  from  the  ingestion  of  more  or  less  highly  contaminated 
meats  are  of  daily  occurrence  in  every  community.  At  each  in- 
stance of  a  death  or  deaths  resulting  from  the  eating  of  bad  meat, 


MEAT  BACTERIA 


153 


the  health  authorities  get  busy  and  almost  invariably  find  the  true 
source  of  the  trouble,  and  there  the  matter  usually  rests.  No  ra- 
tional attempt  is  made  to  prevent  a  repetition  of  the  occurrence. 
Meats  of  all  kinds  when  left  exposed  to  the  air  soon  show  signs 
of  decomposition.  The  aerobic  forms  of  bacteria  are  first  to  de- 
velop, causing  the  decomposition  of  proteids  and  sugars.  Inas- 
much as  sugar  is  usually  present  in  small  amounts  only,  the  sugar 
decomposers  are  soon  crowded  out  by  the  proteid-splitting  forms. 
The  small  amount  of  acid  formed  by  the  sugar  decomposers  is 
neutralized  by  the  ammonia  which  is  formed  during  proteid  de- 
composition. The  aerobes  very  naturally  act  on  the  outside  of  the 
meat  particles,  using  up  the  oxygen  in  the  air  on  and  within  the 
immediate  surface  tissues  of  the  meat.  This  reduction  in  oxygen 
gradually  permits  the  anaerobes  to  get  a  start,  especially  B.  per- 
fringens  and  B.  bifermentens  sporogenes.  These  use  up  proteids 
as  well  as  sugar,  and  the  complete  removal  of  sugar  encourages 
the  more  active  development  of  pure  aerobes  which  act  upon 
proteids  only.     The  following  tabulation  from  the  work  by  Ellis 


Organisms 

Action  on 

Products  Formed 

Proteus  vulgaris 

Proteus  vulgaris 

Streptococcus  longus 

Gluten  and  fibrin 
Casein 

Phenol,  indol,  amines,  fatty  acids. 
Albumoses,  peptones  and  amino- 

Fibrin 

Casein 

Peptone 

Mixture  of  eggs  and 

meat 
Gluten 

Casein 

Albumoses 

acids. 
Tyrosin,  leucin,  amines  and  fatty 

acids. 
Albumoses. 

B.  coli  communis 

B.  coli  communis 

Ammonia  and  indol. 

B.  coli  comnnmis 

Skatol,  phenol,  leucin  and  oxy- 

Micrococcus  pyogenes 

acids. 
Phenol,  indol,  amines   and  fatty 

Aerobic    peptonizing 
acid  bacteria 

lactic 

acids. 
Leucin,  tyrosin,  fatty  acids,  aro- 
matic fatty  acids  and  trypto- 

B. subtUis  and  B.  prodi 

giosus 

phan. 
Leucin,  tyrosin  and  tryptophan. 
Leucin,  tyrosin,  indol,  amines  and 

fatty  acids. 

Cholera  vibrio 

Albumen 

154  BACTERIOLOGICAL   METHODS 

indicates  the  activities  of  putrefactive  bacteria  in  culture,  which 
correspond  with  the  putrefactive  changes  produced  in  nature : 

The  rotting  bacteria  produce  the  familiar  changes  in  meat 
usually  designated  as  spoihng,  rotting  and  tainting,  and  such  meat 
is  universally  recognized  as  unfit  for  food  because  of  the  deleterious 
effects  following  the  ingestion  of  such  meats.  Tainted  meats 
may  appear  entirely  normal  to  the  naked  eye  and  slight  decay  of 
the  inner  tissues  may  not  be  appreciable  to  the  sense  of  smell. 
The  decomposition  changes  resulting  in  the  liberation  of  indol, 
skatol  and  related  substances  having  disagreeable  odors  usually 
begin  near  the  bones  and  joints,  and  such  decomposition  may  not 
become  apparent  until  the  bony  structure  is  exposed  by  cutting 
or  until  the  odors  are  dissipated  more  actively  by  boiling.  This 
odor  is  very  persistent;  boiling  for  several  hours  will  not  cause  it  to 
disappear  entirely.  It  must  not  be  supposed  that  meats  free  from 
bad  odors  are  necessarily  free  from  ptomaines  and  toxins.  For 
example,  perfectly  fresh  meat  may  absorb  these  poisonous  sub- 
stances when  placed  in  contact  with  badly  tainted  meats  and,  again, 
some  toxin-forming  bacteria  do  not  produce  odoriferous  gases. 

The  bacteriology  and  toxicology  of  canned  meats  and  soup 
stocks  containing  meat  has  not  received  the  attention  that  it  should. 
It  is  these  substances  which  are  so  largely  responsible  for  the  mul- 
titudinous lesser  intestinal  disturbances  following  their  use  as  food. 
The  present  methods  of  canning  meats  should  be  thoroughly  in- 
vestigated and  ways  and  means  devised  to  improve  them  in 
accord  with  modern  advance  in  the  manufacture  of  food  products. 
It  is  generally  beheved  that  the  eating  of  canned  meats  and  soups 
is  fraught  with  danger  to  life  and  health,  and  this  is  not  far  from 
the  truth.  The  proper  canning  of  meats  requires  infinitely  more 
care  than  the  canning  of  vegetable  substances.  The  careful  super- 
vision of  the  marketing  of  meats  and  meat  products  is  vastly  more 
important  than  the  supervision  of  vegetable  foods.  It  is  compara- 
tively rare  for  toxins  and  ptomaines  to  be  formed  in  vegetable 
substances,  whereas  this  is  the  rule  in  the  decomposition  of  meats. 


MEAT  BACTERIA 


155 


Furthermore,  meats  decompose  much  more  readily  than  vegetable 
substances,  which  makes  it  necessary  to  observe  greater  care  in  the 
preparation  of  this  class  of  food  for  the  market.  Some  meats 
decompose  much  more  readily  than  others.  Meats  of  higher 
animals  resist  decomposition  longer  than  do  meats  of  lower  ani- 
mals. Fish  meats  decompose  quickly  when  exposed  to  the  air. 
The  story  is  current  among  fishermen  that  certain  kinds  of  fish 


Fig.  48. — Bacillus  welchii,  also  known  as  B.  aerogenes  capsulatus  and  B.  phleg- 
mones  emphysematoscB  in  smear  preparation.  This  is  the  common  "  gas  bacillus, "  be- 
cause of  the  abundant  gas  formation  in  the  tissues  invaded  and  in  culture  media.  _  It 
is  a  plump  large  nonmotile,  anaerobic,  capsulated.  Gram  positive,  spore-bearing 
bacillus  and  is  very  widely  distributed  in  nature. — {Williams.) 

begin  to  decompose  before  they  can  be  removed  from  the  hook. 
This  is  of  course  exaggeration,  but  the  statements  made  indicate 
in  a  way  the  comparative  resisting  power  of  different  kinds  of 
meat  to  rotting  bacteria.  It  is  highly  probable  that  the  difference 
in  the  resisting  power  to  decomposition  is  due  in  part  at  least  to 
the  presence  of  bacteriolysins.  Little  is  known  regarding  the 
changes  which  take  place  in  cold  storage  meats.     Cold  storage 


iS6 


BACTERIOLOGICAL  METHODS 


Fig.  49. — Typical'  cultural 
characteristics  of  Bacillus  aero- 
genes  capsulatus  (B.  welchii)  in 
agar.  Culture  48  hr.  old.  The 
agar  mass  is  separated  by  the  gas 
which  is  formed. — (MacNeal.) 


does  check  the  growth  of  all  kinds  of 
bacteria  and  of  higher  fungi,  but  not 
in  the  same  ratio.  For  example,  the 
freezing  temperature  inhibits  the  de- 
velopment of  the  usual  rotting  bac- 
teria very  effectually,  whereas  many 
of  the  toxin  formers  multiply  slowly, 
in  time  forming  enough  of  the  poison 
to  produce  marked  symptoms  of 
poisoning  when  meat  thus  affected  is 
eaten.  Little  is  known  of  the  changes 
which  take  place  in  incompletely  ster- 
ilized canned  meats,  and  no  attempt 
has  so  far  been  made  to  ascertain  the 
degree  of  decomposition  which  usually 
takes  place  in  the  meats  before  they 
are  placed  in  the  cans  and  sterilized. 
This  is  a  matter  of  the  utmost  impor- 
tance and  should  receive  the  immediate 
attention  of  the  food  bacteriologists. 

What  shall  be  the  routine  method 
in  the  examination  of  meats?  It  is 
quite  evident  that  the  methods  which 
are  applicable  in  the  examination  of 
vegetable  substances  are  not  suitable 
in  the  examination  of  meats.  We 
hereby  suggest  the  following  outline  of 
methods  applicable  in  the  food  labora- 
tory: 

I.  Direct    microscopical    examination    of 
meats. 

a.  Bacteria  on  surface  of  meats. 

b.  Mold  and  spores  present,  as  in  moldy 

bacon,  pork,  etc. 

c.  Presence  of  bladder  worms,  larvae  of 

parasites,  etc. 


EXAMINATION   OF   MEATS  157 

d.  Trichinae  in  pork. 

e.  Cereal  fillers  and  starches  in  sausage  meats.     Tragacanth  fillers. 
/.  Coloring  substances  and  preservatives  in  sausage  meats. 

2.  Plate  cultures.     (Lactose-litmus-agar  and  gelatin  media.) 

a.  Numerical  counts  of  bacteria. 

b.  Number  of  gas  formers  and  acid  formers. 

c.  B.  hotulinus  in  pork  meats. 

3.  Toxicological  tests. 

a.  Inoculation  tests  (guinea-pigs)  to  prove  the  absence  or  presence  of  ptomaines 

or  toxins. 
h.  Tests  for  tuberculous  and  other  diseased  meats. 
4.  Determining  the  source  of  the  meat. 
a.  By  the  precsipitin  test. 
h.  Sugar  test  for  horse  meat. 
c.  Microscopical  identification  based  on  differences  in  the  size  and  structure  of 

the  muscular  fibers  and  the  differences  in  the  size  and  form  of  the  fat  crystals 

derived  from  different  animals. 

Of  the  above  tests  the  numerical  bacterial  count  and  the  toxico- 
logical tests  are  of  the  greatest  importance  and  should  be  carried 
out  in  the  examination  of  suspected  raw  meats,  sausage  meats  and 
of  canned  meats  and  soup  stocks.  There  certainly  should  be  a 
Hmit  to  the  number  of  bacteria  in  all  raw  meats,  whether  ground 
into  sausage  or  not,  as  this  would  be  the  means  of  regulating  the 
sanitary  requirements  in  the  proper  handling  of  meats.  The  only 
practical  method  for  determining  the  quality  of  canned  meats  is  to 
make  inoculation  tests  on  guinea-pigs  or  white  mice,  using  filtered 
aqueous  extracts  of  the  suspected  meat  products.  If  ptomaines 
or  toxins  are  present  the  tests  will  show  it.  It  would  be  very 
desirable  to  work  out  a  micro-chemical  test  for  determining  the 
presence  of  toxins  and  ptomaines  in  meats.  As  above  indicated, 
there  are  some  very  important  differences  between  toxins  and 
ptomaines.  The  former  are  destroyed  by  the  boiHng  temperature, 
whereas  the  latter  are  not.  For  example,  the  thorough  cooking 
of  sausage  meats  prevents  botulism  but  it  does  not  prevent  the 
ill  effects  resulting  from  the  eating  of  meats  with  ptomaine  poison. 
Dried  and  smoked  meats  should  be  examined  for  the  presence 
of  bacteria  and  molds.     Dried    fish  in   particular  is  very  fre- 


158  BACTERIOLOGICAL  METHODS 

quently  highly  contaminated  with  molds.  It  is  very  evident  thai 
the  present  method  of  pickling  fish  of  all  kinds  must  be  changed. 
The  method  of  pickling'^herring,  for  example,  in  wooden  vats  «'^ 
casks  must  be  abandoned,  as  the  containers  are  wholly  unsuital 
from  a  sanitary  standpoint.  The  Hquor  from  canned  fish  (in  1 
cans)  is  frequently  very  highly  contaminated  with  bacteria  m 
spite  of  the  high  salt  content.  The  gelatin  of  the  market  requiri  r 
careful  examination,  as  much  of  the  sheet  variety  is  not  infrequent  I  \ 


Fig.  50.  Fig.  51. 

Fig.  50. — Bacillus  hotulinus  from  a  sugar-gelatin  culture. — {Pittfield,  after  Kolle 
and  Wassermann.) 

Fig.  51. — Bacillus  enteritidis.  Under  this  name  is  included  a  number  of  organ- 
isms of  the  Gaertner  group  which  play  a  very  important  part  in  meat  decomposition 
and  meat  poisoning.  It  is  also  known  as  the  dysentery  group.  The  organisms  are 
actively  motile,  non-sporogenous,  aerobic,  non-liquefying  and  Gram  negative. — 
{Jordan  after  Kolle  and  Wassermann.) 

entirely  permeated  by  mold  and  bacteria,  rendering  it  not  only 
unfit  for  food  for  man  but  also  unsuitable  for  bacteriological  work. 
The  entire  subject  of  meat  poisoning  is  as  yet  not  very  well 
understood.  Dr.  Savage  states  that  the  bacteria  concerned  in 
meat  poisoning  may  be  classed  under  three  groups:  {a)  the  Gaertner 
group  of  bacilli,  {h)  aerobic  bacilli  not  belonging  to  the  Gaertner 
group,  such  as  B.  proteus  and  B.  coli,  and  (c)  Bacillus  hotulinus. 
In  the  majority  of  cases  of  outbreaks  of  fatal  food  poisoning,  some 


EXAMINATION   OF   MEATS  1 59 

rm  of  the  Gaertner  group  of  bacilli  has  been  the  infecting  organ- 
m.  The  Gaertner  bacilli  are  large  coli-typhoid  types  which 
xupy  a  position  intermediate  between  the  chemically  active 
)lon  group  and  the  chemically  inert  typhoid  group,  and  includes 
.  enteritidis,  B.  typhi  murium,  B.  suipestifer  and  B.  paratyphosus 

Sausages  and  ham  are  the  commonest  sources  of  botuHsm. 
rom  the  anaerobic  character  of  the  bacillus  it  follows  that  poison- 
g  is  rarely  due  to  the  eating  of  fresh  sausage  and  pork.  Invasion 
:  meats  by  B.  hotulinus  can  take  place  only  when  the  necessary 
laerobic  conditions  exist,  as  for  example  when  a  ham  is  stored  at 
le  bottom  of  the  pickling  vat  and  entirely  covered  by  the  pickling 
)lution,  and  in  the  interior  of  insufficiently  cooked  sausages  and  in 
:ored  masses  of  sausage  meats. 

The  use  of  the  compound  microscope  in  the  examination  of 
eats  and  meat  preparations  is  still  in  its  infancy.  The  work  done 
lows  very  clearly  that  with  more  experience  very  valuable  in- 
)rmation  can  be  obtained  from  the  microscopical  examination 
^garding  the  quality  of  meats  of  all  kinds.  It  is  highly  probable 
lat  the  microscope  will  show  diagnostic  differences  in  the  muscu- 
,ture  of  different  animals,  thus  making  it  possible  to  determine  the 
Durce  of  the  meat. 

Considerable  attention  has  already^been  given  to  the  micro- 
:opical  study  of  fat  crystals  derived  from  the  fats  of  different 
Decies  of  animals.  So-called  rancid  fats  or  fats  which  have  aged 
onsiderably,  even  though  they  may  not  yet  give  evidence  of  ran- 
Ldity  to  the  unaided  senses,  will  show  more  or  less  abundant 
rystalline  structure,  arranged  in  clusters,  which  may  be  readily 
sen  under  the  low  power  of  the  compound  microscope.  These 
ry stals  are  not  apparent  in  fresh  fats,  but  are  generally  more  or  less 
bundantly  present  in  canned  meats  and  soup  stocks  containing 
nimal  products,  in  meat  extracts  and  in  other  meat  derivatives 
aving  fat  admixtures.  The  presence  of  crystal  clusters  indicates 
at  decomposition  and  these  are  therefore  an  indication  of  the 
quality  of  the  meat  product  containing  them.     The  degree  of  ran- 


l6o  BACTERIOLOGICAL  METHODS 

cidity  or,  to  state  it  more  accurately,  the  quality  of  the  product 
dependent  upon  age  is  in  direct  proportion  to  the  quantity  of  crys- 
talline clusters  present.  It  would  appear  that  the  quantity  of 
crystals  present  is  not  proportional  to  the  amount  of  bacterial 
contamination  and  decomposition.  The  indications  are  that  it  i\ 
possible  to  determine  the  source  of  the  fat  from  the  color,  size  anc 
arrangement  of  the  fat  crystal  aggregates.  For  example,  th( 
crystal  clusters  of  lard  are  smaller  than  those  of  the  fat  of  the  do- 
mestic fowl.  The  fat  crystal  aggregates  of  the  hen  are  compara- 
tively large  and  the  individual  crystals  are  long  and  slender.  The 
inexperienced  analyst  is  apt  to  mistake  the  crystal  clusters  for 
mold  colonies  (Leptothrix) .  This  mistake  can  very  readily  be 
avoided  by  applying  heat  which  causes  the  prompt  melting  of  the 
fat  crystals  whereas  the  mold  hyphae  are  not  greatly  disturbed  or 
changed.  The  differential  characteristics  which  would  be  con- 
cerned in  the  microscopical  examination  of  fat  crystals  may  1 
given  as  follows: 

1.  Differences  in  the  size  of  the  aggregates. 

2.  Differences  in  the  length  of  the  individual  crystals.  ! 

3.  Differences  in  the  diameter  of  the  individual  crystals. 

4.  Differences  in  the  form  of  the  ends  of  the  individual  crystal.  Ends  may  be 
rounded  or  pointed. 

5.  Differences  in  color.  These  will  in  all  probability  pertain  to  different  races  or 
families  of  the  animal  kingdom.  For  example,  lard  crystals  are  colorless  whereas 
those  of  the  domestic  fowl  are  yellowish. 

The  use  of  certain  chemicals  will  aid  in  the  microscopical  find- 
ings. For  example,  sulphuric  acid  produces  characteristic  color 
reactions  with  certain  fats.  If  two  drops  of  concentrated  s\^| 
phuric  acid  are  added  to  twenty  drops  of  goose  fat,  a  greenish-y^ 
low  color  is  produced  which  changes  to  reddish  brown  on  stirrin 
Under  the  same  conditions  cod-liver  oil  turns  a  violet  color  wher 
turtle  oil  turns  brown.  Castor  oil  turns  yellowish  to  yellowis 
brown  and  finally  wine  red  with  a  very  distinct  zone.  A  similar 
reaction  is  observed  with  neats  foot  oil.  Raw  linseed  oil  turns  a 
deep  reddish  brown  to  very  dark  brown.     Lard  oil  shows  a  distinct 


ish 


FAT   CRYSTALS  l6l 

brown  zone  which  deepens  to  wine  red.  The  reaction  for  sperm 
oil  is  much  as  for  cod-liver  oil.  These  color  reactions  with  sul- 
phuric acid  are  perhaps  of  little  value  in  the  detection  of  fat  adul- 
terations and  admixtures  but  they  will  prove  helpful  aids  in  the 
examination  of  these  substances  as  to  identity.  Pure  concen- 
trated acid  should  be  used.  It  must  also  be  kept  in  mind  that  the 
fat  impurities  which  may  be  present  modify  the  color  reactions. 
Pure  samples  of  fats  should  be  kept  on  hand  for  purposes  of  making 
check  and  comparative  tests.  The  Bureau  of  Animal  Industry 
has  suggested  a  method  for  distinguishing  between  fats  and  oils 
derived  from  the  animal  and  the  vegetable  kingdoms  based  upon 
differences  in  the  appearance  of  the  crystals  (phytosterol  and 
cholesterol). 

In  addition  to  the  study  of  fat  crystals  which  are  formed  spon- 
taneously in  more  or  less  decomposed  and  aged  meat  products  as 
above  set  forth,  certain  methods  for  testing  fat  crystals  isolated 
in  the  pure  state  by  chemical  methods  are  now  generally  carried 
out  in  meat  inspection  and  food  laboratories.  These  tests  com- 
bine the  use  of  the  compound  microscope  and  should  therefore 
be  carried  out  by  the  micro-analyst  or  bacteriologist  and  for  that 
reason  are  hereby  included.  R.  H.  Kerr  of  the  Biochemic  Divi- 
sion of  the  Bureau  of  Animal  Industry  has  worked  out  a  method 
for  detecting  vegetable  fats  in  mixtures  of  animal  and  vegetable 
fats  and  vice  versa,  the  method  will  also  serve  to  demonstrate  the 
presence  of  animal  fats  in  supposedly  pure  vegetable  fats.  The 
method  is  a  slight  modification  of  several  methods  which  have  been 
in  use  for  some  time  and  which  are  described  in  various  text-books, 
and  it  is  hereby  given  in  full  as  it  appears  in  Circular  No.  212  (May 
10,  1 9 13)  of  the  Bureau  of  Animal  Industry. 

The  Detection  of  Phytosterol  in  Mixtures  of  Animal  and 

Vegetable  Fats 

Sample. — The  amount  of  sample  used  depends  on  the  amount  of  material  avail- 
able.    From  200  to  300  grams  is  the  amount  usually  taken.     The  test  is  seldom 


1 62  BACTERIOLOGICAL  METHODS 


m3 


attempted  if  less  than  loo  grams  are  available,  and  an  amount  greater  than  500  gra 
is  never  taken. 

Extraction  with  Alcohol. — The  sample  is  melted  and  poured  into  a  flat-bottomed 
flask  of  I -liter  capacity  which  is  closed  with  a  rubber  stopper  perforated  with  three 
holes.  This  flask  is  set  on  the  top  of  the  steam  bath  and  connected  to  a  reflux 
condenser  and  to  a  700  cc.  round-bottomed  flask  containing  500  cc.  of  95  per  cent, 
alcohol.  A  glass  tube  which  is  adjusted  so  that  its  lower  end  is  about  one-fourth  of 
an  inch  above  the  surface  of  the  fat  and  whose  upper  end  is  bent  at  a  right  angle  and 
closed  by  means  of  a  short  piece  of  rubber  tubing  and  a  pinchcock  fills  the  third  hole  in 
the  stopper.  The  distilling  flask  is  set  down  in  the  steam  so  that  the  alcohol  boils 
briskly.  The  outlet  tube  reaches  down  to  the  bottom  of  the  flask  containing  the 
sample  so  that  the  alcohol  vapor  as  it  distills  over  bubbles  up  through  the  fat  and 
keeps  it  in  a  state  of  vigorous  agitation.  The  alcohol  vapor  is  condensed  in  the 
reflux  condenser  and  returned  to  the  flask  containing  the  fat.  The  distillation  is  con- 
tinued until  all  of  the  alcohol  has  collected  in  the  flask  containing  the  fat.  The  dis- 
tilling flask  is  now  disconnected.  The  alcohol  in  the  flask  immediately  ceases  to  boil 
and  soon  separates  from  the  fat.  The  empty  distilling  flask  is  next  connected  to  the 
bent  tube  by  a  piece  of  glass  tubing  of  sufl5cient  length,  the  pinchcock  opened,  and  the 
alcohol  layer  siphoned  off  into  the  distilling  flask.  This  is  then  connected  as  before 
and  the  distillation  continued  until  the  alcohol  has  again  collected  in  the  first  flask. 
It  is  then  siphoned  into  the  distilling  flask  as  before,  and  a  third  extraction  made. 
After  the  third  extraction  the  alcohol  layer  is  again  siphoned  off  into  the  distilling 
flask  and  the  fat  is  discarded.  The  alcohol  now  contains  practically  all  of  the  choles- 
terol and  phytosterol  originally  present  in  the  fat. 

Saponification  and  Extraction  with  Ether. — The  alcohol  in  the  distilling  flask  is 
next  concentrated  by  boiling  to  about  250  cc,  and  20  cc.  of  a  concentrated  potassium- 
hydrate  solution  (100  grams  KOH  dissolved  in  100  cc.  water)  added  to  the  boiling 
liquid.  It  is  boiled  for  10  min.  to  insure  complete  saponification  of  all  the  fat  and 
is  then  removed  from  the  steam  bath  and  allowed  to  cool  almost  to  room  temperature. 
After  it  has  cooled  sufficiently  it  is  poured  into  a  large  separatory  funnel  containing 
500  cc.  of  warm  ether  and  shaken  to  insure  thorough  mixing.  The  mixture  may  be 
clear,  but  is  more  often  opalescent.  There  is  now  poured  in  500  cc.  of  distilled  water, 
and  the  funnel  is  rotated  gently.  Shaking  must  be  avoided,  as  it  leads  to  the  forma- 
tion of  extremely  stubborn  emulsions,  but  the  water  should  be  mixed  with  the  alcohol- 
ether-soap  solution.  Separation  takes  place  at  once  and  is  clear  and  sharp.  The 
soap  solution  is  drawn  off  and  the  ether  layer  washed  with  300  cc.  of  distilled  water, 
shaking  being  still  avoided.  After  this  washing  it  is  washed  repeatedly  with  small 
quantities  of  water  until  all  soap  is  removed.  The  ether  layer  is  then  transferred 
a  flask  and  the  ether  distilled  off.  Distillation  is  stopped  when  the  contents  of 
flask  have  been  reduced  to  about  25  cc,  and  the  concentrated  ether  solution  contai 
ing  the  cholesterol,  phytosterol,  and  all  other  unsaponifiable  matter  is  transferred  to  a 
tall  50  cc.  beaker.  The  evaporation  is  continued  until  all  ether  is  driven  off  and  the 
residue  is  perfectly  dry.  If  desired,  a  tared  beaker  may  be  used  and  the  weight  of  the 
unsaponifiable  matter  determined  at  this  point. 


'^1 


FAT  CRYSTALS 


163 


Preparation  of  the  Acetates. — A  small  amount  (3  to  5  cc.)  of  acetic  anhydrid  is 
added  to  the  dry  residue  in  the  beaker  and  heated  to  boiling  over  a  free  flame,  the 
beaker  being  covered  with  a  watch  glass  during  the  process.  After  a  brief  boiling — a 
few  seconds  is  sufficient — the  flame  is  removed  and  the  beaker  transferred  to  the 
steam  bath  and  left  there  until  the  acetic  anhydrid  is  driven  off. 

Purification  of  the  Acetates. — Thirty-five  cc.  of  hot  80  per  cent,  alcohol  are 
added  to  the  acetylated  residue  in  the  beaker  and  heated  to  boiling  with  vigorous 
stirring.  The  liquid  is  then  filtered  quickly  through  a  folded  filter  and  the  in- 
soluble residue  washed  well  with  boiling  80  per  cent,  alcohol.  The  acetates  of  chol- 
esterol and  phytosterol  are  dissolved,  while  the  greater  portion  of  the  impurities 
present  are  not  dissolved  by  the  alcohol  and  remain  on  the  filter.     Paraffin  and  paraf- 


Phytosterol  crystals. 


Fig.  53. — Cholesterol  crystals. 


fin  oil,  if  present,  are  likewise  separated  by  this  treatment.  The  combined  filtrate 
and  washings  are  next  cooled  to  a  temperature  of  10°  to  12°  C.  and  allowed  to  stand 
at  that  temperature  for  2  to  3  hr.  During  this  time  the  acetates  of  cholesterol 
and  phytosterol  crystallize  from  the  solution.  They  are  removed  by  filtra- 
tion, washed  with  cold  80  per  cent,  alcohol,  and  then  dissolved  on  the  filter  with  a 
stream  of  hot  absolute  alcohol  from  a  wash  bottle,  as  little  alcohol  as  possible  being 
used.  The  alcoholic  solution  of  the  acetates  is  caught  in  a  small  glass  evaporating 
dish,  two  or  three  drops  of  distilled  water  being  added  to  the  solution  and  heat  applied 
if  it  is  not  perfectly  clear.  The  dish  is  then  set  out  on  a  desk  in  the  laboratory  and  the 
alcohol  allowed  to  evaporate  spontaneously.  The  contents  are  stirred  occasionally 
and  the  deposit  of  crystals  which  forms  around  the  edges  of  the  liquid  and  on  the  sides 
of  the  dish  rubbed  down  into  the  solution  with  the  stirring  rod.  As  soon  as  a  good 
deposit  of  crystals  has  formed  they  are  removed  by  filtering  through  a  hardened 


164  BACTERIOLOGICAL   METHODS 

filter,  washed  twice  with  cold  90  per  cent,  alcohol,  and  dried  by  suction.  After 
drying  by  suction  they  are  dried  at  100°  C.  for  half  an  hour  and  the  melting  point 
determined. 

Determination  of  the  Melting  Point. — A  tube  of  about  i  mm.  diam.,  sealed  at 
one  end  and  having  a  slight  flare  at  the  other,  is  filled  to  a  depth  of  about  5  mm.  with 
the  dried  crystals,  which  are  packed  somewhat  firmly  in  the  lower  end  by  tapping  on 
a  hard  surface.  This  is  attached  to  the  bulb  of  a  suitable  thermometer  and  the  melt- 
ing point  determined.  A  thermometer  graduated  from  95°  to  200°  C.  in  one-fifth 
degrees  is  used  in  this  laboratory.  The  determination  is  made  in  an  Anschutz 
apparatus,  the  outer  bulb  being  filled  with  concentrated  sulphuric  acid  and  the  inner 
tube  with  glycerin.  The  apparatus  is  so  adjusted  that  no  correction  of  the  observed 
temperature  is  required.  The  melting  point  of  the  first  crop  of  crystals  usually  gives 
definite  information  as  to  the  presence  or  absence  of  phytosterol,  but  the  conclusion 
indicated  is  confirmed  by  recrystallizing  from  absolute  alcohol  and  again  determining 
the  melting  point.  If  the  crystals  are  pure  cholesterol  acetate,  the  melting  point  of 
the  second  crop  should  agree  closely  with  that  of  the  first.  If  phytosterol  acetate  is 
present,  however,  a  higher  melting  point  should  be  noted,  as  phytosterol  acetate  is 
less  soluble  than  cholesterol  acetate. 


The  Emery  Method  for  the  Detection  of  Beef  Fat  in  Lard 

James  A.  Emery  of  the  Biochemic  Division  of  the  Bureau  of 
Animal  Industry  recommends  the  following  method^  for  detect- 
ing beef  fat  in  lard.  It  is  given  here  because  of  its  value  in  isolat- 
ing the  crystals  of  fats  for  microscopical  examination. 

Technique  of  Method. — Five  grams  of  the  warm  filtered  fat  is  weighed  (on  a  bal- 
ance sensitive  to  o.  i  gram)  in  a  glass-stoppered  graduated  cylinder  of  25  cc.  capacity, 
150  to  1 75  mm.  in  height,  with  an  internal  diameter  of  about  18  mm.,  and  warm  ether 
is  added  until  the  25  cc.  graduation  is  reached.  The  glass  stopper  is  securely  re- 
placed and  the  cylinder  is  shaken  vigorously  until  complete  solution  of  the  fat  takes 
place.  The  cylinder  with  its  contents  is  then  allowed  to  stand  in  a  suitable  place 
where  a  constant  temperature,  at  which  it  is  desired  to  have  the  crystallization  pro- 
ceed, may  be  maintained.  (An  apparatus  described  by  Rogers  proved  efficient  for 
the  maintenance  of  this  constant  temperature.)^     After  18  hr.  the  cylinder  is  re- 

^  Circular  132,  May  23,  1908.  Bureau  of  Animal  Industry,  U.  S.  Dept.  of 
Agriculture. 

2  It  is  necessary  to  observe  great  caution  in  the  use  of  this  form  of  apparatus,  as 
the  sparking  of  the  thermo-regulator  is  a  source  of  danger  if  the  solutions  are  care- 
lessly handled.  A  better  form  for  this  work  would  be  one  in  which  the  temperature 
is  controlled  by  a  circulating  hot- water  system  heated  by  a  small  lamp  outside  of  the 
box,  the  regulation  of  which  could  be  adjusted  by  using  one  of  the  many  forms  of  gas 
regulators  on  the  market. 


FAT   CRYSTALS 


i6s 


moved  and  the  supernatant  ether  solution  carefully  decanted  from  the  crystallized 
glycerids,  which  are  usually  found  in  a  firm  mass  at  the  bottom  of  the  vessel.  Cold 
ether  is  then  added  in  three  portions  of  5  cc.  each  from  a  small  wash  bottle,  care  being 
taken  not  to  break  up  the  deposit  while  washing  and  decanting  the  first  two  portions. 
The  third  portion  is,  however,  actively  agitated  in  the  cylinder  with  a  sharp  rotary 
motion  and  by  a  quick  movement  transferred,  with  the  crystals,  to  a  small  filter  paper. 
The  crystals  are  then  washed  with  successive  small  portions  of  the  cold  ether,  with  the 
use  of  the  wash  bottle,  until  10  to  15  cc.  has  been  used,  dependent  on  the  amount  of 
crystals.  Then  by  means  of  a  slight  exhaust  the 
small  amount  of  remaining  ether  is  rapidly  re- 
moved. The  paper  with  its  contents  is  then 
transferred  to  a  suitable  place,  where  it  should 
be  spread  out  and  any  large  lumps  of  the  glyc- 
erids broken  up  by  gentle  pressure.  When 
dry  the  mass  is  thoroughly  comminuted  and  the 
melting  point  of  the  crystals  determined. 

As  the  difference  between  the  melting  points 
of  the  glycerids  obtained  in  this  manner  from 
beef  fat  and  lard  is  not  very  great,  being  only 
about  3.5  degrees,  and  as  the  writer  has  men- 
tioned a  standard  melting-point  temperature  for 
the  glycerids  of  pure  lard  obtained  under  certain 
conditions,  a  description  of  the  apparatus  used 
in  determining  the  melting  points,  together  with 
its  manipulation,  is  essential  and  may  be  of 
some  assistance. 

Determination  of  the  Melting  Point. — A 
large  test  tube  approximately  150  by  25  mm., 
containing  water  (free  from  air)  into  which  the 
bulb  of  a  thermometer^  with  the  melting-point 
tube  attached  is  immersed,  is  placed  in  a  beaker 

of  water  and  so  adjusted  that  the  surface  of  the  liquid  contained  in  the  two  vessels 
is  at  the  same  level.  The  water  in  the  beaker  should  be  heated  rapidly  to  about 
55°  C.  and  that  temperature  maintained  until  the  thermometer  carrying  the  melt- 
ing-point tube  registers  between  50°  and  55°  C,  then  heat  is  again  applied  and  the 
temperature  of  the  outer  bath  carried  somewhat  rapidly  to  67°  C,  when  the  lamp  is 
removed.  The  melting  point  of  the  crystals  is  regarded  as  that  point  when  the 
fused  substance  becomes  perfectly  clear  and  transparent.  The  use  of  a  dark 
background  placed  about  4  in.  from  the  apparatus  will  prove  of  advantage. 

The  melting-point  tube  should  be  of  about  i  mm.  internal  diam.,  sealed  at  one 
end  and  with  a  slight  flare  at  the  other  extremity,  in  order  that  the  loading  may  be 
expedited.     The  amount  of  the  substance  taken  for  each  determination  should  be 

^  The  thermometer  used  was  one  graduated  in  one-fifth  degrees  and  extending 
from  0°  to  100°  C. 


Fig.  54. — Beef  fat  crystals. 
a,  Clusters  of  crystals  as  seen 
under  the  low  power  of  the 
compound  microscope;  6,  crys- 
tals highly  magnified. 


i66 


BACTERIOLOGICAL   METHODS 


approximately  the  same  and  should  occupy  a  space  about  9  mm.  in  length,  being 
somewhat  firmly  packed  in  the  lower  end  of  the  tube  by  tapping  it  sharply  on  a 
hard  surface.  The  water  in  the  outer  bath  should  be  agitated  frequently  during  the 
determination. 

Possible  Sources  of  Error. — In  applying  the  foregoing  method  too  great  care 
cannot  be  exercised  with  the  preparation  of  the  sample.  The  presence  of  water,  the 
incomplete  solution  of  the  fat  in  the  ether,  or  the  presence  of  small  particles  of 
extraneous  matter  may  interfere  with  the  process  of  crystallization,  frequently  caus- 


FiG.  55. — ^Lard  crystals,  a,  Clus- 
ters of  crystals  as  seen  under  the  low- 
power  of  the  compound  microscope;  b, 
crystals  highly  magnified. 


Fig.  56. — Duck  fat  crystals,  a, 
Clusters  of  crystals  as  seen  under  the 
low  power  of  the  compound  microscope; 
b,  crystals  highly  magnified. 


ing  it  to  proceed  too  rapidly  and  resulting  in  the  formation  of  a  large  mass  of  small 
fluffy  crystals  instead  of  the  compact  mass  of  larger  crystals  desired.  These  fine 
crystals  render  the  preliminary  washing  by  decantation  with  ether  difficult,  and  they 
also  persistently  hold  the  unsaturated  glycerids  in  larger  amount  than  is  desirable. 
The  temperature  at  which  the  crystallization  should  be  allowed  to  proceed  should  not 
be  less  than  15°  C.  nor  more  than  20°  C,  with  the  best  results  obtainable  in  the  neigh- 
borhood of  an  average  between  the  two.  Although  larger  crystals  are  formed  at  the 
higher  temperature  (20°  C),  only  lards  of  high  grade  afford  crystalline  deposits  in 
working  quantity,  and  in  many  cases  where  lards  of  inferior  grades  are  tested  the 
amount  of  solid  glycerids  entering  into  their  composition  is  so  reduced  as  not  to  yield 
any  deposit  at  all. 


HORSE   MEAT  1 67 

Pure  fresh  butter  shows  no  crystalline  structure.  Salted  butter 
will  of  course  show  the  characteristic  salt  crystals.  Melted 
butter  which  is  allowed  to  cool  slowly  shows  a  marked  crystalline 
structure  under  polarized  light,  even  under  the  low  powers  of  the 
compound  microscope,  but  this  is  not  a  diagnostic  character 
inasmuch  as  other  fats  show  a  similar  behavior  with  polarized 
light. 

Horse  meat  has  been  used  as  food  for  man  for  many  ages  and 
is  at  the  present  time  a  regularly  marketed  food  article  in  many 
countries.  During  the  siege  of  Paris  (1870)  when  food  became  very 
scarce,  experiments  with  the  meats  of  various  animals  were  made, 
as  that  of  rats,  mice,  cats,  dogs,  mules  and  horses.  Horse  meat 
especially  met  with  general  favor  and  since  that  time  has  become 
quite  common  in  the  French  meat  markets.  It  is  stated  that  it  is 
a  frequent  substitute  for  beef  in  our  restaurants  (the  cheaper 
eating  places  in  our  larger  cities).  Horse  meat  differs  from  beef 
in  that  it  is  somwhat  coarser  grained,  darker  in  color  and  that 
it  contains  a  higher  percentage  of  glycogen.  As  a  rule  the  meats 
from  cattle  contain  little  or  no  glycogen,  although  it  is  stated  that 
fresh  meat  from  well-nourished  cattle  may  contain  as  much  gly- 
cogen as  does  the  meat  of  the  horse.  It  must  also  be  borne  in 
mind  that  the  meat  from  dogs,  cats,  starved  calves  and  fetuses 
contains  considerable  glycogen.  Should  such  meats  be  added  to 
sausages  the  admixture  might  be  recognized  by  the  color,  the  meat 
of  fetuses  and  starved  calves  being  much  lighter  than  that  of  the 
horse  or  of  mature  cattle. 

In  time  the  glycogen  of  horse  meat  is  changed  into  grape  sugar 
and  will  respond  to  the  Fehling's  solution  reaction  for  sugar. 
For  this  purpose  use  a  cold  aqueous  extract  of  the  suspected  meat. 
In  the  case  of  fresh  horse  meat  the  following  tests  are  recommended. 
The  Brautigam  and  Edelmann  test  for  the  presence  of  horse 
meat  is  made  as  follows:  Grind  or  chop  (finely)  50  grams  of  the 
meat  and  boil  for  i  hr.  in  200  cc.  of  water.  Add  1.5  grams  (3 
per  cent,  by  weight  of  the  meat)  of  caustic  potash  and  heat  over 


1 68  BACTERIOLOGICAL  METHODS 

water  bath  until  the  muscle  fibers  are  disintegrated.  Boil  down  to 
50  grams  and  filter.  When  cool  add  an  equal  part  of  dilute  nitric 
acid  (10  per  cent.)  to  precipitate  the  albuminoids,  and  again  filter. 
Pour  the  filtrate  into  a  test-tube  and  carefully  pour  iodine  water 
down  the  inside  of  the  tube.  If  horse  meat  is  present  a  burgundy 
red  zone  appears  at  the  point  of  contact  of  the  two  solutions.  The 
width  and  intensity  of  the  colored  zone  is  in  direct  proportion  to  the 
amount  of  horse  meat  present. 

If  starch  is  present  (as  in  sausages  and  sausage  meats  seasoned 
with  starch-bearing  spices  or  mixed  with  starch  fillers),  this  must  be 
precipitated  from  the  boiled  meat  extract  and  removed  by  filtra- 
tion. To  the  extract  add  two  or  three  times  the  volume  of  con- 
centrated acetic  acid  and  let  stand  for  2  or  3  hr.,  and  then 
filter  through  two  or  three  thicknesses  of  filter  paper.  Test 
the  filtrate  with  the  iodine  water  as  above  suggested.  However, 
before  making  the  glycogen  test  the  test  for  starch  should  be 
applied,  for  if  it  responds  to  this  test  the  precipitation  of  starch 
must  be  repeated.  Because  of  the  dilution  with  the  three  or  more 
volumes  of  acetic  acid  (to  precipitate  the  starch)  negative  re- 
sults may  be  obtained  in  cases  where  horse  meat  is  present.  It 
is  therefore  advisable  to  precipitate  the  glycogen  by  means  of 
alcohol,  using  from  ten  to  twelve  times  the  volume  of  the  acidu- 
lated meat  filtrate.  The  cloudy  alcoholic  suspension  is  run 
through  a  small  filter  and  the  precipitated  glycogen  on  and  in 
the  filter  paper  is  washed  out  by  means  of  hot  acidulated  (acetic 
acid)  water,  and  this  filtrate  is  then  tested  with  the  iodine  water. 
This  test  is  positive  in  the  presence  of  5  per  cent,  quantities  of 
horse  meat.  The  wine-red  color  reaction  is  temporary  only  and 
it  must  be  kept  in  mind  that  dextrin  interferes  with  the  reaction. 

Because  of  the  fact  that  meats  other  than  that  derived  from 
the  horse  may  contain  glycogen,  it  is  sometimes  necessary  to 
supplement  the  above  color  reaction  with  the  biological  test  or 
the  precipitin  test  which  has  come  into  use  within  recent  years. 
The  general  routine  for  making  the  test  is  as  follows:     Inject 


PRECIPITATION  TEST  169 

(subcutaneously  or  intravenously)  rabbits  with  10  cc.  of  filtered 
defibrinated  horse  blood  (or  serum)  every  other  day  five  or  six 
times.  At  the  end  of  this  time  draw  blood  from  the  rabbit, 
allow  it  to  clot  kept  on  ice,  remove  the  serum  and  filter,  where- 
upon the  reagent  is  ready  for  use.  Express  and  extract  (in  saline 
solution)  the  juice  from  the  meat  suspected  to  contain  horse  meat, 
filter  and  keep  on  ice  until  wanted  for  use.  To  the  filtrate  thus 
prepared  add  a  few  drops  of  the  equinized  rabbit  serum.  If 
cloudiness  and  sHght  whitish  precipitate  forms  it  constitutes  a 
positive  test,  proving  conclusively  that  the  suspected  meat  is 
horse  meat  or  contains  horse  meat.  Only  raw  fresh  meat  re- 
sponds to  this  test.  Heating  destroys  the  action  of  the  reagent. 
Inoculating  rabbits  with  the  defibrinated  and  filtered  blood 
serum  of  various  animals,  as  of  hog,  domestic  fowl,  deer,  dog, 
bear,  etc.,  and  testing  in  the  manner  outlined  in  the  following 
method  by  Dr.  Karl  F.  Meyer  of  the  State  University  of  Cali- 
fornia, the  meat  of  the  responding  animal  may  be  identified. 

The  Precipitin  Test  for  the  Detection  of  Horse  and  Deer 
Meat  and  for  Meat  Adulterations  in   General 

The  method  can  be  used  for  fresh,  dried,  frozen,  pickled,  raw 
and  smoked,  but  not  for  boiled,  meat.  The  meat  may  not  be 
heated  above  6o°-7o°  C.  for  the  biologic  test. 

For  the  tests  are  needed: 

a.  Specific  antisera  (anti-horse  or  deer  precipitin  serum;  pre- 
cipitin) . 

h.  Aqueous  extract  of  the  meat  to  be  identified  (precipitinogen). 

I.  Antisera. — The  sera  must  be  specific  and  highly  active 
against  the  meat  protein  to  be  determined.  Rabbits  are  in- 
jected subcutaneously,  intravenously  or  intraperitoneally  with 
serum,  defibrinated  blood  or  extract  of  the  fat  free  meat.  The 
best  results  are  obtained  by  inoculating  fresh  serum  intravenously. 
The  sera  for  injection  can  readily  be  obtained   from  abattoirs 


170  BACTERIOLOGICAL   METHODS 

or  from  serum  institutes  or  laboratories.  Horse  serum  is  not  as 
toxic  to  rabbits  as  are  some  other  sera.  Meat  extracts  should 
always  be  filtered  to  avoid  infection  of  the  animals  to  be  im- 
munized, but  extensive  slouching  is  likely  to  occur  with  any 
method  of  immunization  and  the  mortality  rate  is  high.  The 
blood  or  serum  used  as  antigen  can  be  preserved  by  the  addition 
of  chloroform  (1-2  per  cent.),  or  by  drying. 

On  account  of  the  individual  differences  existing  in  rabbits 
in  regard  to  the  development  of  precipitins,  it  is  advisable  to 
treat  at  least  six  animals  at  the  same  time.  The  injections  of 
2-3  cc.  of  horse  or  deer  serum  are  made  at  intervals  of  5  days. 
Ten  days  after  the  last  injection  the  blood  is  tested  for  pre- 
cipitins. The  further  treatment  of  the  animals  differs  individu- 
ally, depending  on  the  precipitin  contents  of  the  rabbits.  Ani- 
mals which  show  a  high  precipitin  reaction  are  given  subsequent 
inoculations  subcutaneously  or  intraperitoneally,  to  avoid  ana- 
phylactic death  which  frequently  results  from  intravenous  in- 
oculations. Some  rabbits  fail  to  produce  precipitins,  whatever 
the  method  used. 

Fornet  and  Muller^  recommend  the  intraperitoneal  injection 
of  5,  10  and  15  cc,  respectively,  of  protein  material  on  the  ist, 
2d  and  3d  day,  respectively.  The  test  for  antibodies  is  carried  out 
on  the  12th  day.  Gay  and  Fitzgerald^  inject  on  three  consecutive 
days  I  cc.  of  the  antigen,  bleed,  and  test  the  serum  on  the  loth 
day.  Both  methods  frequently  give  very  good  results.  The 
precipitin  content  of  an  immune  serum  is  occasionally  titrated 
during  the  process  of  immunization  by  withdrawing  a  few  cubic 
centimeters  of  blood  from  an  ear  vein.  The  hair  over  the  marginal 
vein  is  removed  and  the  skin  rubbed  with  alcohol.  A  fine  pipette  is 
introduced  into  the  vein  and  the  blood  collected  by  capillary  attrac- 
tion or  by  suction.  It  is,  however,  advisable  for  the  beginner  to 
cut  the  vein  transversely  and  to  collect  the  blood  in  a  centrifugal 
tube.     The  hemorrhage  is  stopped  by  covering  the  wound  with 

^  University  of  California  publications,  Pathology,  Vol.  II,  75,  191 2. 


PRECIPITATION   TEST  171 

cotton  soaked  in  liq.  ferri  sesquichloridi  (ferric  chloride)  or  by 
placing  a  small  hemostat  for  3^  to  i  hr.  on  the  incision.  The 
serum  which  has  separated  from  the  clot  is  centrifugalized  and 
the  titer  is  determined  as  follows: 

Preliminary  Titration. — Into  each  of  a  series  of  six  test-tubes 
place  2.0  cc.  of  the  following  dilutions  of  serum  (horse  or  deer) 
antigen,  mixed  with  0.85  per  cent,  saline  1:100,  1:500,  i:iooq, 
1 :  5000,  1 :  10,000  and  i :  20,000.  To  each  cubic  centimeters  of  the 
dilution  o.i  cc.  of  antiserum  is  added.  The  solution  of  1:1000 
should  become  turbid  instantaneously  or  within  i  to  2  min.,  the 
other  dilutions  in  from  3  to  5  min.  The  serum  should  have  a 
titer  of  1:20,000;  that  means  the  serum  should  cause  a  turbidity 
in  a  dilution  (of  horse  serum  or  extract  of  meat)  of  i :  20,000  in  less 
than  5  min.  The  antiserum  is  either  introduced  by  allowing 
it  to  run  down  the  side  of  the  tube  (no  shaking  is  permissible), 
or  it  is  stratified  on  the  diluted  horse  serum.  In  the  first  case  the 
turbidity  appears  from  the  bottom,  in  the  second  case  in  form  of  a 
grayish  ring;  both  reactions  are  positive.  The  coloration  is 
best  seen  against  a  dark  background.  The  pipettes  and  test- 
tubes  must  be  perfectly  clean  and  sterile.  The  equipment  de- 
signed by  Uhlenhuth  is  very  satisfactory.  The  test-tubes  are 
long  and  narrow,  10  cm.  by  0.8  cm.,  and  are  suspended  in  beveled 
holes  of  the  test-tube  rack.  Pipettes  of  i  cc.  capacity  graduated 
into  Koo  cc,  and  5  and  10  cc.  pipettes  graduated  into  J^o  cc. 
will  be  found  satisfactory. 

Preservation  of  Serum. — In  case  the  titer  of  the  serum  is 
satisfactory,  the  rabbit  is  bled  to  death  (aseptically)  from  the 
carotids.  For  full  details  on  technique,  consult  the  text-books  on 
Immunity.  The  centrifuged  serum  should  be  perfectly  clear 
and  sterile  and  should  not  be  opalescent.  Kept  cool  and  in  the 
dark  (ice  chest)  it  will  remain  potent  for  months,  even  years. 
To  avoid  opalescence  the  animal  should  be  bled  only  after  a  period 
of  fasting.  On  account  of  autoprecipitation,  it  will  lose  some  of 
its  potency.     The  precipitate  formed  can  be  removed  by  cen- 


172  BACTERIOLOGICAL  METHODS 

trifugalizing  or  by  filtration,  but  the  titer  must  again  be  tested. 
Preservatives  such  as  carbolic  acid,  etc.,  should  not  be  added  to 
the  sera.  Sterile  sera  are  obtained  by  filtration  through  Berke- 
feld  filters.  Drying  of  the  sera  on  filter  paper  is  the  best  method 
known  for  preserving  them  {Jacohsthal  und  v.  Elsler). 

2.  The  Preparation  of  the  Meat  Extract. — To  make  the  bio- 
logic test  for  horse  or  deer  meat,  remove  from  the  deeper  parts 
of  the  specimen,  by  means  of  a  flamed  or  boiled  knife  and  through 
a  fresh  opening,  a  piece  of  muscle  of  about  30  grams  weight. 
It  should  contain  as  little  fat  as  possible.  On  a  sterilized  tile 
(best  covered  with  unused  writing  paper)  chop  the  meat  carefully. 
The  finely  minced  meat  is  placed  in  a  sterilized  100  cc.  Erlenmeyer 
flask  and  spread  out  with  a  sterile  glass  rod  and  covered  with 
50  cc.  sterile  saline  solution.  Salted  meat  is  washed  for  10 
min.  in  a  large  flask  with  distilled  water,  renewing  the  water 
several  times,  without  shaking  the  flask. 

The  mixture  of  saline  and  meat  is  kept  for  about  6  hr. 
at  room  temperature,  or  over  night  in  the  refrigerator.  To 
obtain  a  clear  solution  the  flask  should  not  be  shaken. 

Since  the  presence  of  fat  interferes  with  the  reaction,  it  is 
advisable  to  remove  it  by  means  of  ether  and  chloroform.  To 
make  the  extraction,  take  75-100  grams  of  the  minced  meat, 
place  in  a  large  Erlenmeyer  flask  and  cover  with  equal  parts  of 
ether  and  chloroform.  After  24  hr.  the  ether  and  chloroform  are 
poured  off,  the  meat  is  washed  once  or  twice  with  sahne  solution 
and  then  extracted,  as  stated  above. 

To  determine  whether  a  sufficient  quantity  of  protein  sub- 
stances has  passed  into  solution,  place  2  cc.  of  the  extract  in  a 
test-tube  and  shake  vigorously.  If  a  fine  foam  develops  and 
persists  for  some  time,  the  extraction  may  be  said  to  be  sufficiently 
complete.  The  protein  solution  must  be  perfectly  clear  and  must 
therefore  be  filtered.  With  extracts  from  fresh  meat  this  is 
usually  accomplished  by  filtering  through  a  firm  filter  paper 
previously  moistened  with  saline  solution.     If  it  is  not  crystal 


PRECIPITATION   TEST  1 73 

:]ear,  and  especially  if  the  meat  to  be  examined  was  fat  or  salt, 
t  is  filtered  through  a  sterile  Berkefeld  or  through  a  layer  of 
nfusorial  earth  stratified  in  a  Btichner  funnel. 

The  filtrate  is  suitable  for  the  test  when  a  foam  is  developed 
)y  shaking  and  when  it  contains  about  i  part  of  protein  in  300 
)arts  of  salt  solution.  To  determine  this,  2  cc.  of  the  clear  filtrate 
ire  placed  in  a  test-tube  and  heated,  and  a  drop  of  dilute  nitric 
icid  (sp.  gr.  1. 1 53)  is  added;  if  a  marked  cloudiness  and  a 
iocculent  precipitate  forms,  the  extract  is  too  highly  concentrated 
md  must  be  diluted  with  normal  salt  solution  until  the  heat  and 
icid  test  causes  only  a  diffuse,  opalescent  cloudiness  which  settles 
:o  the  bottom  of  the  tube  after  5  min.  as  a  slight  precipitate. 

Before  proceeding  with  the  test,  the  reaction  of  the  meat  ex- 
Tact  should  be  tested  with  litmus  paper  and  if  it  is  found  to  be 
icid  it  should  be  neutralized  very  carefully  with  o.i  per  cent, 
odium  hydroxide  or  magnesium  oxide  solution.  Only  slightly 
icid  or  alkahne  solutions  should  be  used.  For  the  extraction  of 
the  meat,  spigot,  tap  or  distilled  water  should  not  be  used.  Fresh 
neat  frequently  produces  a  sufficiently  strong  protein  solution 
n  I  hr.  In  boiled,  preserved  and  decomposed  meat,  the  ex- 
traction proceeds  very  slowly  (24  hr.)  and  the  solutions  are 
iifiicult  to  clarify. 

Technique  of  the  Test. — If,  for  example,  the  object  is  to 
determine  whether  a  piece  of  meat  is  horse  flesh  or,  if  sausage, 
contains  the  meat  of  this  animal,  the  test  is  conducted  as  follows: 

Tube  I. — 2  cc.  of  unknown  extract  (i  :  300)  +0.1  cc.  of  anti-horse  serum. 
Tube  2. — 2  cc.  of  unknown  extract  (i  :  300)  -{-  o.i  cc.  of  normal  rabbit  serum. 
Tube  3. — 2  cc.  of  horse  flesh  extract  (i :  300)  +  0.1  cc.  of  anti-horse  serum. 
Tube  4. — 2  cc.  of  pork  extract  (i  :30o)  -|-  0.1  cc.  of  anti-horse  serum. 
Tube  5. — 2  cc.  of  beef  extract  (i :  300)  H-  0.1  cc.  of  anti-horse  serum. 
Tube  6. — 2  cc.  of  saline  solution  -f  0.1  cc.  of  anti-horse  serum. 

The  immune  serum  is  added  to  each  tube  very  carefully  and 
run  down  the  sides  of  the  tube,  or  stratified.  The  tubes  must 
not  be  shaken.     The  tubes  are  kept  at  room  temperature.     The 


174 


BACTERIOLOGICAL  METHODS 


test  must  not  be  made  with  a  mixture  of  the  sera  of  differc 
rabbits. 

Interpretation  of  the  Results. — If  in  tubes  i  and  3  a  misty  clou 
ness  should  appear  within  5  min.,  and  if  a  definite  precipitate  fori 
within  30  min.,  the  other  tubes  remaining  perfectly  clear,  1 
extract  is  very  probably  one  of  horse  flesh  or  the  flesh  of  soi 
other  single-toed  animal.  Precipitates  which  develop  more  slo^^ 
cannot  be  considered  as  positive.  The  protein  of  horses  and 
donkeys  cannot  be  differentiated  by  this  test.  In  a  similar  mn- 
ner,  tests  may  be  made  for  the  meat  of  deer,  dogs  or  any  ot] 
animals,  if  the  respective  immune  sera  are  used  with  the  extrri 


Fig.  57. — Types  of  syringes:     i,  Roux's  bacteriologic  syringe;  2,  Koch  syrin 
3,  Meyer's  bacteriologic  syringe.     The  Meyer  syringe  is  the  simplest  and  best  t<  1 
general  purposes. — {McFarland.) 

Heterologous  precipitates,  which  occur  when  antisera  are 
added  to  concentrated  foreign  protein  solutions,  rarely  are 
disturbing  factors  of  the  tests  when  the  above  technique  is  used. 
The  elective  absorption  (according  to  Kister  and  Weichardt) 
with  the  foreign  protein  is  occasionally  necessary  for  scientific 
tests. 

The  organoleptic  tests  are  not  always  conclusive  as  to  the 
quality  of  the  meat.  It  is  a  well-known  fact  that  the  stinking  or 
putrefactive  odors  are  generally  wholly  absent  in  even  highly 
decayed  salted  and  brine-pickled  fish  and  meats  and  in  heavily 
seasoned  sausage  meats  and  in  smoked  meats.     On  the  othen 


MEAT  BACTERIA  175 

md,  it  is  advisable  to  reject  or  condemn  all  meats  which  emit 
Jensive  odors,  provided  such  odors  are  not  normal  to  the  meat, 
nder  normal  offensive  odors  may  be  mentioned  the  fishy  odor 
:  meats  from  animals  which  feed  upon  fish,  mussels  and  other 
:iuatic  animals;  the  sex  odor  which  is  often  marked  in  the  meats 
om  older  males;  the  various  vegetable  odors  due  to  feeding, 
ich  as  the  turnip  odor  and  taste  in  beef,  fenugreek  odor,  etc., 
:c.  Distinctively  putrefactive  odors  in  meats  are  a  very  reliable 
idication  of  their  unfitness  for  consumption.  Marked  changes 
L  consistency  (sloppy,  smeary  and  porous  meats)  and  in  color 
grayish,  yellowish,  greenish)  usually  indicate  advanced  stages  of 
^composition.  Some  authorities  have  recommended  that  the 
resence  of  free  ammonia  should  be  the  test  for  putrefactive 
langes  in  meats  and  should  serve  as  the  basis  for  condemnation 
rocedures,  but  others  point  out  the  fact  that  toxins  are  formed 
/en  before  there  is  any  appreciable  formation  of  ammonia.  The 
ifcst  guide  to  the  quality  of  meats  is  undoubtedly  the  bacterio- 
•gical  test.  As  to  the  question  on  what  bacteriological  findings 
lall  the  quality  estimates  of  meat  be  based,  it  is  suggested  that 
idgment  be  based  upon  the  number  of  bacteria  present  and 
^nerally  irrespective  of  kind.  If  exposed  and  comminuted  meats 
0  not  contain  more  than  1,000,000  bacteria  per  gram,  they  may  be 
resumed  to  be  reasonably  wholesome.  The  exceptions  to  this 
umerical  limit  are  the  finding  of  pathogenic  and  toxin-forming 
acteria.  The  conclusive  proof  of  the  mere  presence  in  meats 
f  bacteria  which  are  pathogenic  to  man  is  su£&cient  to  condemn 
ich  meats.  It  is  reasonable  to  assume  that  most  bacterial  in- 
asions  of  meats  are  of  the  putrefactive  kind  and  hence  objection- 
ble,  and  it  is  therefore  fair  and  just  to  all  concerned  to  fix  a  nu- 
lerical  limit  at  which  such  foods  are  still  reasonably  wholesome, 
s  suggested.  There  are,  ha;v^ever,  those  notable  exceptions  where 
leat  contains  toxins  and  ptomaines  in  quantities  sufficient  to 
roduce  serious  and  even  fatal  poisoning  without  bacteria  being 
resent,  as  when  fresh  meat  has  been  in  contact  with  decomposed 


176 


BACTERIOLOGICAL  METHODS 


and  toxin-bearing  meats  from  which  it  has  taken  up  the  poisons 
by  absorption.  It  is  therefore  desirable  and  often  necessary 
to  supplement  the  bacterial  count  by  the  toxicity  test. 

The  numerical  limit  above  suggested  (1,000,000  per  gram 
of  the  meat  substance)  pertains  to  bacteria  found  upon  the  ex- 
terior of  the  meat  bulk  or  in  the  outside  cells  and  tissues  of  the 
meat  bulk  or  meat  particles.     Proper  care  must  therefore  be 

observed  in  taking  samples 
and  in  preparing  the  sample 
for  plating.  In  the  case  of 
bulk  meats  such  as  whole 
slaughtered  animals,  hams, 
bacon,  etc.,  pieces  as  nearly 
cubical  as  possible  (about  i 
gram  each)  are  removed  with 
a  sharp  sterilized  scalpel, 
the  outer  surface  of  the  meat 
forming  one  face  of  the  cube. 
This  is  to  be  weighed  and 
pulped  in  a  sterile  mortar 
with  an  equal  amount  of 
sterile  normal  salt  solution 
and  this  pulped  material  is 
then  made  up  to  the  desired 
dilutions  for  plating,  using 
normal  salt  solution.  Gela- 
tin media  should  be  used  for 
culturing  and  incubation  should  be  done  at  20°  C.  for  a  period  of 
3  days  and  the  counts  made.  In  the  case  of  sausage  meats  and 
comminuted  meats  generally,  take  i  gram  quantities,  pulp 
thoroughly  and  mix  thoroughly  with  the  required  amount  of  nor- 
mal saline  and  plate.  In  the  case  of  soups  and  soup  stocks  hav- 
ing a  meat  or  meat  derivative  base,  take  i  cc.  quantities,  from 
the  thoroughly  mixed  sample,  dilute  and  plate. 


Fig.  58. — Illustrating  the  method  of 
making  an  intravenous  injection  into  a 
rabbit.  The  ear  is  manipulated  to  induce 
hyperaemia  and  the  surface  vein  is  com- 
pressed near  the  base  of  the  ear,  to  facilitate 
the  inserting  of  the  syringe  needle. — 
{McFarland.) 


TOXINS   IN   MEAT  1 77 

Weinzirl  and  Newton  describe  a  method  of  determining  the 
bacterial  content  of  meat,  in  which  the  meat  is  ground  in  a  mortar 
with  sterile  sand  and  normal  salt  solution  to  obtain  an  emul- 
sion for  inoculation  into  the  culture  media,  and  report  the  appli- 
cation of  this  method  to  the  determination  of  the  bacterial  con- 
tent of  a  number  of  samples  of  market  Hamburger  steak.  The 
result  showed  that  the  standard  of  1,000,000  bacteria  per  gram 
advocated  as  a  maximum  limit  for  the  salable  product  is  much 
too  low,  as  nearly  all  the  samples  examined  would  be  condemned 
Dn  this  basis,  though  showing  no  taint  or  other  evidences  of 
putrefaction.  The  authors  propose  a  limit  of  10,000,000  bacteria 
3er  gram. 

For  making  toxicity  tests  of  meats,  broths,  sausage  meats, 
50up  stocks  and  other  meat  products,  the  following  general 
nethod  is  recommended.  In  case  of  solids  such  as  meats  (raw, 
smoked,  cooked,  canned  or  pickled),  sausages,  sausage  meats,  etc., 
CO  grams  of  a  well-mixed  average  sample  are  well  pulped  in  10 
:c.  of  boiled  distilled  water.  Let  stand  for  20  min.  with  frequent 
stirring.  Express  and  filter  the  extract  through  a  clay  bougie, 
rhe  toxins  being  soluble  will  be  found  in  the  filtrate.  Inject 
>  cc.  of  the  clear  filtrate  into  the  subdermal  connective  tissue 
)r  intraperitoneally  into  guinea-pigs  or  white  mice,  using  three 
mimals  for  each  test.  If  one  or  more  of  the  animals  thus 
noculated  die  within  48  hr.,  or  if  they  show  marked  symptoms 
)f  intoxication  without  dying,  the  meat  is  unfit  for  consumption, 
n  the  case  of  soups,  broths,  soup  stocks,  chop  suey  and  other 
neat  products  which  contain  liquid,  the  procedure  is  much  simpler. 
Fake  suitable  quantities  of  the  thoroughly  mixed  sample  and 
liter,  first  through  filter  paper  and  finally  through  the  clay 
30ugie,  as  for  the  meat  extract  already  described,  and  inject 
\  cc.  quantities  as  already  explained.  The  toxicity  tests  should 
n  all  cases  be  supplemented  by  the  plate  count. 

Botulism  or  sausage  poisoning  is  due  to  a  toxin  (botulin) 
ormed  by  the  Bacillus  hotulinus   (Lat.,   hotulus,  a  sausage),  a 


178  BACTERIOLOGICAL   METHODS 

large  anaerobic  sporogenous  saprophyte  especially  common  in 
sausages  and  sausage  meats,  particularly  in  liver  sausages,  blood 
sausages,  jelly  sausages,  in  hams,  in  liver  pate,  canned  meats, 
etc.,  etc.  The  bacillus,  inclusive  of  the  spores  and  the  highly 
virulent  toxins  which  it  forms,  are  destroyed  by  boiling  and 
thorough  cooking.  The  digestive  ferments  do  not  destroy  the 
toxin.  The  usual  smoking  of  hams  and  sausages  does  not  de- 
stroy the  toxin  or  the"  bacillus.  The  bacillus  is  killed  by  strong 
brines,  but  this  does  not  also  destroy  the  toxin.  The  oval  spores 
are  quite  readily  killed  by  heat  and  chemicals.  Heating  to  80° 
C.  for  I  hr.  kills  them.  Ichthyotoxism  (fish  poisoning)  and 
mytilotoxism  (shellfish  poisoning)  are  closely  akin  to  botulism 
and  are  in  all  probability  caused  by  the  same  bacillus  or  perhaps 
a  varietal  form  of  B.  botulinus.  The  occurrence  of  the  Bacillus 
bolulinus  is,  however,  not  limited  to  pork  and  sausage  meats. 
Well-authenticated  cases  are  on  record  of  the  occurrence  of  this 
bacillus  in  canned  vegetables  and  in  domestically  prepared  string 
beans  served  without  previous  heating.  There  is  no  doubt 
that  the  heat  employed  in  the  canning  process  destroys  the  toxin 
formed,  but  the  temperature  may  not  always  be  high  enough  to 
kill  all  of  the  bacilli  and  their  spores  even  though  the  spores  are 
not  very  resistant  to  heat  (80°  C).  Bacillus  botulinus  does  not 
multiply  in  the  living  organism.  It  grows  readily  in  slightly 
alkaline  media  at  a  temperature  of  18°  to  25°  C.  At  higher  tem- 
peratures (35°  to  37°  C.)  it  grows  only  sparingly  and  without 
the  formation  of  toxin.  Cultures  give  out  an  odor  of  butyric 
acid. 

In  pickled,  canned  and  otherwise  prepared  and  preserved 
meats,  and  mixtures  of  meat  and  vegetables  (chop  suey,  pork  and 
beans,  etc.),  the  processes  of  bacterial  development  are  greatly 
modified.  The  use  of  deodorants,  of  preservatives  and  color- 
ing agents  mask  or  obscure  many  of  the  decomposition  changes 
in  meats.  Very  frequently  the  only  cause  for  suspicion  is  an 
unusually  heightened  color  or  a  lack  of  the  normal  meat  flavor. 


MEAT  BACTERIA  179 

Sausage  meats  are  found  on  the  market  so  highly  colored  as  to 
produce  a  red  ink  with  the  water  in  which  they  are  boiled.  The 
meat  dealer  tries  to  deceive  the  housewife  by  stating  that  the 
red  color  is  derived  from  the  rich  red  blood  of  the  meat  itself, 
whereas  the  red  coloring  matter  of  the  blood  is  decomposed  by 
the  boiling  and  the  boiled  meat  extract  is  only  slightly  colored. 
Very  frequently  pickled  pigs'  feet  appear  on  the  market  which  look 
quite  normal,  the  only  suspicious  character  being  an  unusual 
pallor  of  the  surface  with  a  smeary  consistency  and  a  lack  in  the 
flavor.  On  microscopical  examination  it  will  be  found  that  the 
surface  of  the  meat  is  covered  or  coated  with  yeast  cells,  mold 
hyphse  and  mold  spores  and  bacteria.  The  American  method 
of  making  sausage  and  sausage  meats  from  carelessly  and  pro- 
miscuously handled  meat  trimmings  which  accumulate  during 
the  day's  work  in  the  retail  meat  markets,  is  accountable  for  the 
high  contamination  with  bacteria  and  other  organisms  (10,000,000 
to  100,000,000  per  gram).  Such  sausage  meats  are  also  very 
frequently  colored  to  reduce  the  pallor  due  to  the  use  of  ex- 
cessive amounts  of  fatty  tissue  trimmings,  thus  leading  the  cus- 
tomer to  believe  that  there  is  a  considerable  amount  of  muscular 
(red  meat)  tissue  present.  The  coloring  also  serves  to  hide  the 
beginnings  of  decomposition  changes  in  the  meat.  Preservatives 
are  added  to  check  and  mask  the  decomposition  changes  which 
have  begun  to  manifest  themselves.  It  is  unlawful  to  add 
coloring  substances  to  sausage  meats,  but  it  is  permissible  to  color 
sausage  casings. 

Numerous  chemical  tests  for  ascertaining  the  existence  of 
putrefactive  changes  in  meats  have  been  recommended.  The 
Ebers  test  appears  to  have  met  with  considerable  favor  and  is 
made  as  follows:  Into  a  test-tube  pour  about  3  cc.  of  a  mixture 
composed  of  i  part  of  pure  hydrochloric  acid,  i  part  ether  and 
3  parts  alcohol.  This  tube  may  be  closed  with  a  perforated 
rubber  stopper  carrying  a  glass  rod  which  is  pushed  through 
the  opening  of  the  stopper  so  that  the  end  almost  touches  the 
13 


i8o 


BACTERIOLOGICAL  METHODS 


liquid  in  the  tube.  Dip  the  free  end  of  the  tube  into  the  meat 
pulp,  meat  extract  or  meat  broth  and,  after  shaking  the  tube  in 
order  to  fill  it  with  the  acid  vapors,  insert  the  rod,  closing  the  tube 
with  the  rubber  stopper.  If  the  juice  or  the  meat  particle  is 
from  decayed  meat,  a  grayish  smoky  vapor  appears  at  the  end 
of  the  glass  rod,  which  settles  to  the  surface  of  the  Hquid.  There 
must  be  no  free  ammonia  in  the  room  while  making  the  test. 

The  test  is  not  applicable  to 
pickled  meats.  This  test 
should  be  made  supplementary 
to  the  microscopical,  bacterio- 
logical and  toxicological  exami- 
nations already  explained.  In 
place  of  the  test-tube  or 
reagent  glass  above  recom- 
mended, the  small  perfume 
sample  bottles  with  glass  rod 
stoppers  may  be  used  in  mak- 
ing the  test. 

Fig.  sg.— Bacillus  tetani  as  seen  in  a         Sausage    meat    binders    or 
S'gS/To;  sTif  for^Ton°U1i:   fiU^s  are  very  readily  detected 
The  pale  globules  are    by    means    of    the   compound 
(X  looo). — (Kolleand         .  r^  ^       ^  j 

microscope.     Corn  starch  and 

wheat  starch  fillers  are  most 
commonly  employed,  the  object  in  adding  them  being  to  increase 
the  water  content  of  the  sausage  meat.  Some  brands  of  sausage 
contain  corn  meal  and  other  cereal  products.  Egg  albumen  and 
tragacanth  fillers  are  used  occasionally,  and  it  is  said  that  it  is 
possible  to  increase  the  water  content  of  the  meats  by  30  per 
cent,  with  only  3  per  cent,  of  the  tragacanth  filler.  The  increase 
in  water  content  through  the  use  of  the  starch  fillers  is  about  5 
to  10  per  cent.  In  examining  meats  for  starch  fillers  or  added 
cereal  it  must  not  be  forgotten  that  some  of  the  spices  used 
contain  starch  (pepper,  allspice). 


others  do  not. 
blood  corpuscles. 
Wasserman.) 


CEREAL  IN  SAUSAGE  MEAT  l8l 

Graham,  of  the  laboratory  division  of  the  Bureau  of  Animal 
Industry,  has  recommended  a  method  for  determining  the  per- 
centage of  starch  added  to  sausages  and  sausage  meats.  A 
small  pellet  of  a  thoroughly  mixed  sample  of  the  meat  preparation 
is  well  pulped  and  teased  out.  Make  the  usual  slide  mount, 
using  just  enough  of  the  prepared  material  to  fill  the  space  be- 
tween slide  and  cover,  using  some  pressure.  Count  the  number 
of  starch  granules  in  the  areas  (squares)  of  the  ocular  scale  and 
compare  with  the  known  number  of  similar  starch  granules  in 
I,  2,  3  and  4  per  cent,  mixtures  of  the  same  starch.  Rarely  does 
the  amount  of  starch  filler  added  exceed  3  or  4  per  cent.  Mr. 
Graham  states  that  the  method  gives  results  accurate  within  10 
per  cent.,  which  is  sufficiently  accurate  fo.r  all  practical  purposes. 

It  is  suggested  that  the  special  spore  and  mold  counter  de- 
scribed elsewhere  (A  or  B,  Fig.  5)  be  used  with  the  ocular  counting 
scale  (Whipple's)  for  making  the  starch  determinations  in  sausage 
meats.  The  exact  number  of  starch  granules  in  mixtures  con- 
taining I  per  cent,  of  starch  should_,be  carefully  ascertained,  follow- 
ing the  general  method  recommended  for  finding  the  number  of 
oil  globules  representing  i  per  cent,  of  butter  fat  in  milk.  For 
determining  the  number  of  granules  in  i  per  cent,  suspensions  of 
the  starch,  it  is  suggested  that  weak  solutions  of  gum  arable  (i  per 
cent.)  be  used.  The  gum  solution  keeps  the  meat  particles  as  well 
as  the  starch  granules  in  suspension  until  the  counting  is  com- 
pleted. Having  once  determined  the  exact  number  of  granules 
in  I  per  cent,  of  the  starch  suspension,  it  is  a  simple  matter  to 
make  comparative  determinations  of  homologous  starch  in 
sausage  meats,  or  in  other  substances,  as  may  be  required. 

Add  I  gram  of  a  well-mixed  sample  of  the  sausage  or  sausage 
meat  to  about  2  cc.  of  water  in  a  suitable  dish  and  mix  thor- 
oughly, in  order  to  wash  the  starch  from  the  meat  particles.  Next 
add  enough  of  the  gum  arable  solution  to  make  a  total  of  9  cc. 
of  the  liquid,  thus  making  a  dilution  of  i-io.  Mix  thoroughly 
in  order  that  the  starch  present  in  the  meat  may  be  uniformly 


1 82  BACTERIOLOGICAL  METHODS 

distributed  and  make  the  counts  as  for  spores  or  yeast  cells,  and 
from  the  findings  determine  the  percentage  of  starch  which  has 
been  added.  This  quantitative  method  for  determining  added 
starch  is  applicable  even  if  the  starch  has  been  dextrinized  through 
the  cooking  of  the  sausages,  provided  the  individual  granules 
are  still  recognizable  and  provided  also  the  identity  of  the  starch 
is  still  ascertainable.  Corn  meal  and  corn  starch  are  the  more 
common  sausage  fillers  used  in  the  United  States. 

The  above  method  for  determining  the  percentages  of  starch 
in  mixtures  could  also  be  employed,  modified  to  suit  special  cases, 
in  the  examination  of  compounds  of  flour,  of  meals,  for  ascertain- 
ing the  percentage  of  starch  in  baking  powders,  in  almond  meal, 
in  adulterated  mustard  and  in  other  products  where  starch  or  flour 
is  used  for  purposes  of  adulteration,  and  to  ascertain  the  pro- 
portions in  flour  or  meal  compounds,  etc. 

In  frozen  meats  the  red  blood  corpuscles  are  almost  com- 
pletely decolorized  and  disintegrated  (hemolyzed),  changes 
which  are  readily  observed  under  the  compound  microscope. 
The  microscope  will  also  prove  useful  in  the  detection  of  added 
coloring  substances.  The  micro-sublimation  test  will  readily 
demonstrate  the  presence  of  benzoic  and  salicylic  acids  in  meats 
and  meat  products. 

The  microphytic  examinations  of  meat  include  the  following 
groups  of  the  plant  kingdom : 

1.  Penicilliuin  Species.^ — Especially  common  on  hams,  bacon 
and  smoked  meats  generally.  These  molds  are  essentially  aerobic 
saprophytes  and  are  therefore  found  on  the  exterior  of  meats. 

2.  Aspergillus  Species. — These  molds  are  apt  to  occur  on 
and  in  fish  meats,  in  gelatin,  in  canned  meats  and  in  pickled 
meats. 

3.  Mucor  Species. — These  small  molds  are  less  common 
than  the  above.  They  may  occur  on  pickled  meats  and  on 
meats  that  are  kept  in  damp  places. 

4.  Yeasts. — Yeast   cells   may   occur   on  pickled   meats  and, 


MEAT   BACTERIA 


183 


more  especially,  in  meats  and  meat  products  which  contain  starch 
and  sugar. 

5.  Bacteria. — It  is  not  necessary  to  enter  into  any  extensive 
discussion  of  the  different  species  and  varieties  of  bacteria  which 
may  occur  in  and  upon  meats.  The  more  important  bacterial 
invasions  of  meats  have  already  been  mentioned.  The  following 
is  a  partial  list  of  the  more  important  species  which  the  food  bac- 
teriologist may  be  called  upon  to  look  for  in  meats: 

a.  Bacillus  hotulinus. — Most  common  in  sausages,  as  already 


/ 

x-A 

\''  '^^ 

\ 

'      I 

^^^. 

^ 

Fig.  60. — B.  tetani,  showing  flagellae. 


stated   elsewhere.     Forms   highly  virulent   toxins   and  produces 
rancid  changes. 

b.  Bacillus  tuberculosis. — Will  be  found  in  meats  of  tuber- 
culous animals. 

c.  Bacillus  tetani. — May  occur  in  meat  products,  more  es- 
pecially in  gelatin.  It  is  essentially  anaerobic  but  thrives  better 
in  association  with  aerobes,  and  it  produces  one  of  the  most 
virulent  toxins  known  which  is,  however,  very  unstable  in  its 
chemical  composition  and  easily  destroyed.  A  temperature  of 
60°  to  65°  C.  destroys  it  and  it  is  also  very  quickly  destroyed  on 
exposure  to  air  and  light.  The  danger  from  the  tetanus  bacillus 
pertains  to  possible  inoculation  with  the  bacillus  rather  than  the 


i84 


BACTERIOLOGICAL  METHODS 


ingestion  of  the  toxins,  which  might  be  formed  outside  of  the 
body  and  absorbed  by  the  meat. 

d.  Cadaver  bacilli. — Under  this  head  are  included  a  variety 

of  bacteria  which  cause  putrefactive 
changes  in  dead  animals  and  in  meats, 
with  toxin  and  ptomaine  formation, 
and  to  which  reference  has  already 
been  made. 

e.  Bacillus  anthracis. — The  anthrax 
bacillus  may  occur  in  all  food-produc- 
ing animals,  and  its  isolation  from 
beef  and  other  meats  may  become  an 
occasional  necessity  in  the  food  labo- 
ratory. 
^  /.    Staphylococcus     group. — These 

I  '  may  occur  in  great  abundance  in  hv- 

I  ^  ing  animals,  causing  septic  decomposi- 

tion changes  in  tissues  and  organs. 
1%^^.  ^'  ^l^^plococcus    group.— Like   the 

F  mM.^  Staphylococci,    these    organisms  pro- 

duce pyemic  or  septic  changes  in  liv- 
ing animals. 

h.  Numerous  other  bacteria  may 
on  occasion  come  to  the  attention  of 
the  food  bacteriologist,  as  the  bacillus 

Fig.    6i.-Tetanus    bacillus    ^^   ^^S  cholera,   of  swine  plague,  of 

swine  erysipelas  and  others.     In  this 
connection  we   must   not   forget   the 
possible  presence  in  beef,  and  less  fre- 
quently also  in  pork,  sheep  and  horses,  of  the  ray  fungus  {Actin- 
omyces  hovis)   which  is  the  primary   cause  of  "lumpy  jaw"  in 
cattle  and  which  disease  is  transmissible  to  man. 

Examination  of  meats  for  the  presence  of  encysted  trichina; 
{Trichinella  spiralis)  is  incidental  rather  than  a  routine  in  the 


stab  culture  in  glucose-gelatin  6 
days  o\±~{McFarland,  after 
Fraenkel  and  Peifer.) 


TRICHINA 


185 


food  laboratory.  Even  if  the  meat  is  found  to  contain  trichinae 
it  does  not  warrant  condemnation  procedures,  because  these  organ- 
isms are  harmless  provided  the  meat  is  properly  cooked  be- 
fore eating;  however,  it  cannot  be  denied  that  no  consumer  could 
be  persuaded  to  use  meat  thus  infected.  The  examination  of 
pork  for  the  presence  of  encysted  trichinae  was  at  one  time  a 
regular  routine  in  the  larger  slaughtering  houses  of  America  be- 
cause of  the  European  (largely  German)  boycott  against  Ameri- 


FiG.  62. — Actinomyces  bovis  from  broth  culture  (X  1000). — (Williams.) 


can  pork.     In  recent  years  the  routine  examination  for  trichinae 
has  been  very  generally  abandoned. 

Trichinae  are  not  uniformly  distributed  in  the  muscular 
tissue  of  the  animal.  They  are  most  abundant  in  the  diaphragm, 
next  in  the  base  of  the  tongue,  in  the  laryngeal,  lumbar,  mas- 
ticatory, and  abdominal  muscles  and  nearest  the  tendinous 
insertions  of  the  bones.  They  are  never  found  in  adipose  tis- 
sue. They  may  occur  in  wild  hogs,  in  dogs  and  in  bears  and  of 
course  also  in  man.     To  examine  meat  for  trichinae,  cut  bits 


1 86  BACTERIOLOGICAL   METHODS 

from   the   organs   of   chief   distribution   of   the  parasite.     From 
these  samples  cut  small  fiat  pieces  and  compress  between  two 


Fig.  63. — Colony  of  Actinomyces  bovis  from  cow. — (Williams.) 

glass  slips  and  examine  under  the  low  power  of  the  compound 
microscope.     As  a  clearing  agent  a  solution  of  acetic  acid  (1-30) 


Fig.  64. — Encysted  Trichina  spiralis  (Trichinella  spiralis)  in  muscle  tissue. — 

(Stilt,  after  Ziegler.) 

may  be  used.     To  clear  sections  of  salted  hams  or  other  meat, 
use  diluted  potassium  or  sodium  hydrate.     Examining  minced 


EGGS  187 

meats  and  sausages  for  trichinae  requires  greater  care  and 
persistency. 

Encysted  trichinae  retain  their  vitality  for  a  long  period 
of  time  when  kept  at  a  low  temperature,  and  persist  even  after 
the  meat  has  undergone  decomposition  through  bacterial  infec- 
tion. The  wandering  embryos  are  harmless  and  the  muscle 
trichinae  continue  their  development  only  in  another  host,  as 
man,  dog  or  bear.  In  the  intestinal  tract  of  this  second  host 
they  become  sexually  matured,  growing  to  a  length  of  0.5  to  0.75 
mm.,  and  produce  young  in  large  numbers.  Trichinella  does  not 
produce  ova. 

The  inexperienced  analyst  might  mistake  vinegar  eels  (in 
pickled  meats),  Miescher's  bodies  (Sarcocystis),  lime  concretions, 
muscle  degenerations  and  tri chinas-like  worms  \Fseudo-trichince) , 
found  in  the  muscles  of  the  rat,  mouse,  rabbit,  fowl,  fish,  mole 
and  other  animals,  for  trichinae. 

18.  The  Bacteriological  Examination  of  Eggs  and  Egg 
Products 

Among  the  foods  which  require  the  attention  of  the  bac- 
teriologist are  eggs  and  egg  products  such  as  evaporated  eggs, 
frozen  eggs  and  dried  egg  albumen.  Many  fresh  eggs  are  quite 
free  from  bacteria,  or  if  bacteria  are  present  they  do  not  exceed 
negligible  quantities,  usually  not  over  500,000  per  cc.  Ex- 
tensive investigations  made  by  Stiles  (Bureau  of  Chemistry) 
show  that  the  contamination  of  eggs  is  in  proportion  to  age  and 
favorable  temperature.  Thus  during  warm  weather  the  bacterial 
development  is  quite  rapid,  whereas  cold  retards  such  develop- 
ment. Placing  contaminated  eggs  in  cold  storage  checks  bacterial 
development  temporarily  and  even  causes  a  reduction  in  the 
Qumber  of  organisms  present  at  the  time  the  eggs  were  placed 
in  storage,  but  within  a  short  time  the  temporary  numerical 
reduction  in  bacteria  is  not  only  regained  but  there  is  a  steady 


l88  BACTERIOLOGICAL   METHODS 

increase  in  proportion  to  the  time  of  storage,  until  a  maximum 
development  is  reached.  The  bacterial  flora  of  the  white  and 
of  the  yolk  of  the  egg  differs  quantitatively  as  well  as  qualita- 
tively. It  may  happen  that  the  yolk  is  badly  infected  while 
the  white  is  in  comparatively  good  condition.  As  a  rule,  how- 
ever, if  the  yolk  is  highly  contaminated  the  white  is  similarly 
affected.  In  fact  the  first  decomposition  changes  generally  take 
place  in  the  periphery  of  the  egg  albumen,  the  infection  taking 
place  via  the  exterior  of  the  shell. 

Commercially,  eggs  are  designated  as  fresh,  stale,  storage; 
firsts,  seconds  and  thirds  (when  sorted  as  to  size);  watery  and 
weak  when  the  white  is  thin;  heat  eggs;  leakers,  checks  and 
mashed  when  the  shell  is  more  or  less  broken;  eggy,  strong, 
musty,  sour  and  stale  as  to  odor;  blood  ring,  sour  rot,  white  rot, 
light  rot,  spot  rots,  moldy,  black  rots,  etc.,  when  more  or  less 
rotted  and  decomposed;  green  or  grass  eggs  when  the  white  is 
more  or  less  green  colored  through  the  invasion  of  bacteria. 
These  terms  have  no  scientific  importance  and  are  of  no  signifi- 
cance to  the  food  bacteriologist,  beyond  that  of  indicating  the 
probable  or  likely  condition  and  contamination  and  probable 
cause  of  the  change  or  deterioration  of  the  eggs  so  designated. 

The  old-time  popular  methods  of  testing  eggs  by  candling, 
by  shaking  to  determine  ''looseness,"  floating  on  brine,  noting 
discoloration  of  the  shell,  and  by  the  odor,  have  their  value  in 
practice  but  are  far  from  reliable.  An  egg  which  gives  off  the 
odor  of  sulphuretted  hydrogen  is  universally  recognized  as  bad, 
rotten  or  spoiled.  In  Germany  eggs  are  pronounced  spoiled  if  the 
white  is  gelatinous  in  consistency  (as  in  old  eggs  from  which 
moisture  has  escaped)  or  yellowish  in  color  (also* due  to  age), 
or  if  the  yolk  is  more  or  less  adherent  to  the  shell  or  is  more  or 
less  mixed  with  the  white.  A  fresh  egg  broken  in  the  manner! 
customary  in  the  kitchen  allows  the  entire  contents,  yolk  and 
all,  to  fall  out  into  a  receptacle  without  rupturing  the  yolk. 
The  white  should  be  of  uniform  consistency,  uniformly  trans-" 


EGGS 


189 


lucent  and  without  marked  yellowish  or  amber  coloration.  The 
yolk  should  be  uniformly  soft  and  entirely  free  from  all  lumpiness 
and  should  not  be  adherent  to  the  shell. 

Eggs  are  preferably  used  in  the  comparatively  fresh  state, 
that  is,  within  a  few  days  or  at  the  longest  8  days  after  they  are 
laid.  It  is,  however,  not  always  possible  or  practicable  to  use 
the  eggs  \yhile  still  fresh,  and  egg  preservation  has  become  a  very 
important  industry.  Eggs  may  be  preserved  in  brine,  in  liquid 
glass  and  in  various  chemical  preservatives.  They  may  also 
be  preserved  in  oil,  in  lard,  or  coated  with  tallow,  wax  or  paraffin, 


Fig.  65. — Egg  membrane  as  seen  under  the  high  power  of  the  compound 
microscope  (X  450). 

in  order  to  keep  out  air  bacteria  and  molds  and  also  to  check  the 
evaporation  of  moisture  from  the  interior.  There  is  an  opinion 
among  some  poultrymen  that  eggs  will  keep  much  longer  if 
placed  in  a  definite  position  (vertically  with  narrower  end  down). 
The  now  generally  employed  and  preferred  method  for  preserv- 
ing eggs  is  to  keep  them  in  storage  at  a  temperature  as  low  as  it 
is  possible  to  make  it.  This  is  perhaps  the  simplest  and 
cheapest  method  for  keeping  eggs  in  the  natural  state.  However, 
as  already  stated,  cold  storage  eggs  gradually  deteriorate,  through 
bacterial  invasion  and  through  loss  of  moisture,  in  direct  ratio  to 


I  go  BACTERIOLOGICAL  METHODS 

time,  until  they  finally  become  unsuitable  for  consumption.  If 
fresh-laid  eggs  were  thoroughly  cleansed  and  sterilized  externally, 
coated  with  sterilized  wax,  tallow,  paraffin  or  placed  in  liquid 
glass  and  then  put  in  cold  storage,  they  would  no  doubt  remain 
wholesome  for  a  period  of  5  months  to  i  year.  Under  the 
usual  conditions,  cold  storage  eggs  show  marked  deterioration 
in  the  course  of  2  or  3  months  I'as  indicated  by  loss  of 
moisture,  yellowing  of  the  albumen,  softening  of  the  yolk,  loosen- 
ing, increase  in  size  of  the  air  chamber  and  by  the  increase  in  the 
bacterial  count.  The  increase  in  the  size  of  the  air  chamber  is 
due  to  the  shrinkage  of  the  egg  mass,  resulting  from  the  loss  of 
moisture.  According  to  Greenlee,^  the  loss  in  weight  of  eggs 
is  due  to  evaporation  of  moisture  to  the  external  atmosphere  but 
the  decrease  in  moisture  of  the  white  is  not  wholly  due  to  external 
evaporation,  as  the  yolk  takes  up  a  part  of  the  moisture,  thus 
increasing  the  moisture  and  weight  of  the  yolk  and  also  account- 
ing for  the  increased  liquidity  and  explaining  the  tendency  on  the 
part  of  the  yolk  to  rupture  and  the  white  to  gelatinize.  Fresh 
eggs  break  well,  whereas  old  eggs,  including  those  kept  in  storage, 
break  badly  as  a  rule.  The  egg  mass  does  not  leave  the  shell 
readily,  the  yolk  may  be  adherent  to  the  shell,  likewise  the  white, 
and  the  yolk  membrane  ruptures  easily  and  the  result  is  generally 
a  mess. 

Evaporated,  dried  and  frozen  eggs  have  come  into  extensive 
use  in  recent  years.  For  these  purposes,  the  cheapest  and  hence 
the  poorest  market  eggs  are  generally  employed.  There  is  indeed 
an  attempt  made  to  cull  the  bad  eggs  at  the  factory,  but  this  is, 
as  a  rule,  not  done  in  an  efficient  manner.  The  eggs  are  usually 
broken  by  women  and  the  egg  mass  is  thoroughly  mixed  and 
dried  by  spraying  into  a  drying  chamber  or  by  spreading  on  a 
drying  belt,  or  the  drying  may  be  done  in  very  shallow  pans. 
Of  course  the  temperature  must  be  kept  below  the  coagulation 

^  Deterioration  of  Eggs  as  shown  by  Changes  in  the  Moisture  Content.     Circular 
8Si  Food  Research  Laboratory,  Bureau  of  Chemistry,  Aug.  20,  191 1. 


EGGS  191 

point  of  the  albumen.  Instead  of  drying,  the  egg  mass  may  be 
preserved  by  freezing  and  keeping  it  frozen  until  wanted  for 
use.  The  important  factors  are  the  use  of  fresh  wholesome  eggs 
and  cleanliness.  Pennington^  summarizes  the  importance  of 
cleanliness  as  follows:  "The  preparation  of  frozen  and  dried 
eggs  parallels  the  milk  problem.  In  dairying  it  is  first  necessary 
to  obtain  a  cow  giving  good  milk.  Then  her  products  must  be  so 
handled  that  it  is  maintained  in  good  condition  until  it  reaches 
the  consumer,  a  question  that  has  engaged  the  attention  of 
sanitarians  for  many  years  and  is  still 

the  subject  of  study.     The  hen  seems  O     ^  ^  O 

to  be  more  reliable  as  a  producer  of  ^  o  S^oO  ^ 

good  eggs  than  is  the  cow  of  good        cd  o  q  ^<0    S^^ 
milk.     In  either  case  the  ignorance        ^     o   ^Oo  ^(So  ^ 
or  carelessness  of  man  results  in  the      9^  8       j;)^      ^^h'^  &A 
addition    of  multitudes  of  bacteria     ^opQ^^n   ^    rs 
which  will,  and  frequently  do,  spoil     OvO      O  ^  ^  r,  (^      ^ 
the  product  for  food  purposes.     The         0^8  ^^cP^    ^  ^ 
fundamental  in  the  handling  of  whole-  ^ 

some  milk  is  cleanliness.  The  fun-  '''''■  ,t-^^TsZl,7~ '" 
damental  in  the  handling  of  good  eggs 

is  also  cleanliness,  a  cleanliness  based  upon  and  adapted  to  the 
work  to  be  accomplished." 

Recently  (19 13-19 14),  American  poultrymen  of  the  Pacific 
Coast  have  raised  a  hue  and  cry  against  the  importation  of  stor- 
age eggs  from  China,  Analysts  in  food  laboratories  have  been 
called  upon  to  examine  these  as  to  their  suitableness  for  eating 
and  cooking  purposes.  Barring  differences  incidental  to  acci- 
dents in  shipping,  the  Chinese  storage  eggs  compare  favorably  in 
quahty  with  those  cornered  by  the  American  egg  trust  or  combine. 

What  is  needed  in  food  laboratories  is  a  method  for  determining 
when  an  egg  is  or  is  not  suitable  for  human  consumption.     Ac- 

^  Practical  Suggestions  for  the  Preparation  of  Frozen  and  Dried  Eggs.  Circular 
No.  98,  Bureau  of  Chemistry,  July  31,  1912. 


192  BACTERIOLOGICAL  METHODS 

cording  to  observations  made,  it  would  appear  that  the  direct 
microscopical  examination  of  the  white  of  the  egg  will  give  this 
information.  The  yolk  of  the  egg  does  not  lend  itself  to  direct 
examination  because  of  the  fat  globules  (cholesterin)  and  proteid 
granules  present  which  interfere  with  the  observation  of  the 
bacteria.  The  procedure  as  carried  out  in  the  laboratories  of 
the  California  College  of  Pharmacy  is  to  break  the  egg  into  a 
suitable  sterilized  dish,  after  having  washed,  dried  and  flamed  the 
egg  thoroughly.  The  egg  mass  is  carefully  tilted  and  poured 
from  one  portion  of  the  shell  into  the  other  until  most  of  the 
white  is  separated  from  the  yolk.  Mix  the  white  thoroughly 
by  means  of  a  sterilized  egg  beater  and  examine  under  the  com- 
pound microscope,  making  the  counts  with  the  hemacytometer. 
Fresh  eggs  contain  bacteria  in  such  small  numbers  as  to  make 
counting  difficult.  The  principal  organism  found  in  the  white  of 
the  egg  is  a  coccus  form,  of  fairly  large  size,  having  some  of  the 
characters  of  a  diplococcus  combined  with  those  of  yeasts.  Mul- 
tiplication appears  to  be  by  a  modified  budding  process.  After 
the  cell  has  developed  to  maturity  it  sends  out  a  second  cell 
which  at  first  appears  as  a  scarcely  perceptible  speck  or  protuber- 
ance elevated  above  the  surface.  This  protuberance  grows 
larger  and  larger  until  it  has  the  dimensions  of  the  mother  cell, 
whereupon  the  two  cells  separate.  A  chain  of  three  cells  is 
not  uncommon  and  chains  of  fours  and  even  fives  may  be  found. 
At  first  these  structures  were  believed  to  be  proteid  or  perhaps 
lecithin  particles,  and  in  fact  attempts  to  cultivate  them  in  arti- 
ficial media  resulted  in  failure.  There  is,  however,  little  doubt 
that  they  are  micro-organisms  which  develop  preferably  in  the 
white  of  the  egg.  They  apparently  do  not  increase  in  large 
numbers.  The  highest  number  recorded  in  cold  storage  eggs  was 
about  180,000,000  per  cc.  They  appear  to  increase  in  direct 
ratio  to  the  age  of  the  egg.  They  do  not  stain  very  readily.  The 
most  satisfactory  stain  appears  to  be  carbol-fuchsin,  though 
the  organisms  are  not  in  the  least  acid  fast.     They  do  not  stain 


EGGS  193 

with  methylene  blue.  This  egg  albumen  organism  requires 
further  careful  study. 

The  following  general  methods  for  making  bacteriological 
examinations  of  eggs  and  of  egg  products  are  recommended. 

To  examine  fresh  eggs  for  bacteria  proceed  as  follows:  Scrub 
the  egg  well  in  clean  steriHzed  water  by  means  of  a  sterile  hand 
brush.  Soak  in  corrosive  sublimate  solution  (i-iooo)  for  3 
min.,  rinse  in  boiled  water  and  wipe  dry  with  a  sterilized 
cotton  cloth.  Flame  the  end  to  be  opened  and  set  into  a  suitable 
holder  (the  ordinary  breakfast  table  egg  holder  will  answer  the 
purpose  after  being  sterilized),  and  with  a  sterile  instrument 
crack  open  the  flamed  end  and  by  means  of  a  sterile  forceps 
pick  away  small  pieces  of  the  shell  without  rupturing  the  egg 
membrane,  making  a  hole  large  enough  to  introduce  a  sterile 
pipette.  Rupture  the  egg  membrane  with  a  sterile  forceps  and 
take  out  2  cc.  of  the  w^hite  of  the  egg,  place  in  a  tared  flask  with 
broken  glass  and  reweigh.  Add  10  cc.  of  physiological  salt  solu- 
tion and  shake  for  10  min.,  and  then  plate  definite  volumes. 

To  plate  the  yolk,  break  the  egg  in  the  usual  manner  merely 
being  careful  that  the  yolk  membrane  is  not  ruptured;  remove 
the  white  of  the  egg  by  pouring  back  and  forth  in  the  two  parts  of 
the  shell.  Let  the  yolk  rest  in  the  larger  part  of  the  shell  and 
puncture  the  vitelHne  membrane  by  means  of  a  sterile  forceps. 
Withdraw  2  cc.  of  the  yolk  and  proceed  as  for  the  white  of  the 

egg. 

To  make  bacterial  counts  of  eggs  which  are  quite  badly 
spoiled  (rotten  eggs),  simply  break  the  thoroughly  cleansed  egg 
in  the  usual  manner  into  a  suitable  steriHzed  dish  and  mix  thor- 
oughly (white  and  yolk)  by  means  of  a  sterile  egg  beater.  Let 
stand  for  5  min.,  suitably  covered  to  keep  out  air  bacteria. 
Skim  off  the  foam  caused  by  the  stirring  and  take  up  i  cc.  of  the 
mixed  egg  mass  and  add  to  9  cc.  of  boiled  distilled  water,  and 
shake  for  10  min.  as  for  the  examination  of  the  white  of  fresh 
eggs.     Make  direct  counts  from  suitable  dilutions  and  also  plate 


194  BACTERIOLOGICAL  METHODS 

definite  quantities  (dilutions  as  i-io  and  i-ioo).  Certain  rotting 
bacteria  attack  eggs  very  readily.  As  all  housewives  know, 
eggs  which  are  broken  become  unfit  for  use  in  a  very  short  period 
of  time,  because  of  decomposition  changes.  The  dried  eggs  of 
the  market  are  very  likely  to  show  high  bacterial  counts  and  the 
manufacture  of  evaporated  eggs,  dried  egg  albumen  and  other 
egg  products  intended  for  use  as  food  should  be  carried  on  under 
suitable  methods,  keeping  in  mind  the  speedy  decomposition  of 
the  egg  material. 

Dried  and  evaporated  eggs  and  dried  egg  albumen  are  ex- 
amined for  bacteria  by  the  direct  method  and  also  by  the  plating 
method. 

It  is  certainly  evident  that  no  complicated  bacteriological 
testing  is  necessary  to  determine  the  unfitness  of  a  rotten  egg  or 
an  egg  which  is  highly  musty  or  discolored  as  shown  by  the 
candle  test.  The  important  problem  in  estimating  the  significance 
of  rotten  eggs  is  what  percentage  of  rotten  eggs  may  be  present 
in  an  acceptable  lot  or  shipment?  It  is  evident  that,  under  ordi- 
nary conditions,  the  bacterial  count  of  eggs  not  sufficiently 
spoiled  to  be  noticeable  to  the  unaided  senses  (eggs  taken  from  a 
lot  in  which  there  are  numerous  rotten  eggs)  will  exceed  many 
milHons  per  cc.  Condemnation  of  eggs  for  human  consumption 
should  not  be  based  upon  the  percentage  of  rotten  eggs  present, 
but  rather  upon  the  finding  of  a  given  number  of  bacteria  in  a 
mixed  sample  of  the  whites  of  one  dozen  average  eggs  taken  from 
the  lot,  exclusive  of  completely  rotted  eggs.  Eighteen  eggs 
are  taken  from  the  lot,  cleaned  as  already  suggested,  the  eggs 
broken  one  by  one,  pouring  the  white  of  each  egg  into  a  suitable 
container  and  rejecting  all  eggs  in  which  the  white  cannot  be 
separated  from  the  yolk  without  mixing.  If  six  or  more  out  of 
the  eighteen  eggs  are  decidedly  bad,  the  lot  is  to  be  condemned 
without  further  examination. 

If  twelve  out  of  the  eighteen  eggs  break  in  such  a  manner 
as  to  make  it  possible  to  separate  the  whites  from  the  yolks, 


EGGS  195 

then  the  whites  are  to  be  thoroughly  mixed  and  the  bacterial 
count  made  in  the  manner  already  explained.  It  is  suggested 
that  if  the  bacterial  count  (inclusive  of  the  coccus  form  and  motile 
forms  or  any  other  recognizable  forms)  exceeds  200,000,000 
per  cc.  the  eggs  are  not  suitable  for  human  consumption.  In 
certain  instances  the  limiting  count  should  no  doubt  be  lower. 
The  analyst  should  take  into  consideration  some  of  the  other 
factors  indicative  of  the  quality  of  the  eggs,  as  gelatinous  con- 
dition of  the  whites,  yellowing  of  the  whites,  tendency  to  adhere 
to  the  shell,  etc.  There  is  the  possible  occurrence  of  highly  con- 
taminated yolks  with  the  white  in  passable  condition.  This  is, 
however,  a  condition  not  likely  to  occur  in  the  entire  dozen 
selected  for  the  count  and  may  be  ignored  as  a  factor  having  any 
practical  value  in  the  rating  of  eggs  as  to  quality. 

In  case  the  direct  examination  gives  doubtful  results,  it  is 
recommended  that  the  plating  method  be  resorted  to  for  check 
purposes.  For  culturing  it  is  advised  that  egg  albumen  be  used 
for  the  bacteria  in  the  white  of  the  egg  and  yolk  media  for  the 
yolk  bacteria.  In  all  media  for  egg  bacteria  egg  peptone  should 
be  used  instead  of  the  ordinary  meat  peptone.  The  following 
media  will  be  found  useful : 

Whole  Egg  Medium 
Contents  of  one  egg 

Egg  albumen  peptone  (Merck's) i  gram 

Distilled  water 100  cc. 

Mix  ingredients  thoroughly  in  a  sterile  container  by  means 
3f  a  sterilized  egg  beater.  Titrate  to  +  i.oo.  Filter  through 
cotton.  Tube  and  plate  as  may  be  desired.  Coagulate  care- 
fully and  sterilize  as  for  culture  media  in  general. 

This  medium  is  recommended  for  making  plate  counts  of 
3gg  bacteria,  of  the  yolk  as  well  as  those  of  the  white  of  the  egg. 
[t  will  also  be  found  an  excellent  medium  for  culturing  the  tubercle 
bacillus.  For  plating  the  egg  albumen  the  following  medium  is 
recommended : 
14 


196  BACTERIOLOGICAL  METHODS 

Egg  Albumen  Medium 
Whites  of  two  eggs 

Egg  albumen  peptone  (Merck's) i  gram 

Distilled  water 100  cc. 

Prepare  as  for  whole  egg  medium.  If  egg  yolk  bacteria  are 
to  be  cultured,  the  following  medium  may  be  used : 

Egg  Yolk  Medium 
Yolk  of  two  eggs 

Egg  albumen  peptone  (Merck's) i  gram 

Distilled  water 100  cc. 

Several  investigators  have  reported  toxins  in  eggs.  It  is 
also  known  that  some  persons  are  peculiarly  susceptible  to 
eggs,  being  more  or  less  injuriously  affected  on  eating  even 
perfectly  fresh  eggs.  This  phenomenon  is  by  some  ascribed  to 
personal  idiosyncrasy  and  others  suggest  that  this  is  due  to 
toxins  present  to  which  certain  persons  are  perhaps  peculiarly 
susceptible.  The  poisonous  principles  present  in  eggs  should  be 
more  carefully  investigated.  The  possibility  of  toxin  forma- 
tion in  cold  storage  eggs  also  requires  further  careful  study. 

Eggs  decompose  very  rapidly  when  the  shell  and  membrane 
are  broken  and  soon  become  unfit  for  use,  due  to  bacterial  de- 
velopment. The  shell  of  the  egg  serves  to  prevent,  or  at  least  to 
check,  for  a  time  the  development  of  the  egg-rotting  bacteria 
through  exclusion  of  oxygen  (of  the  air).  It  is,  however,  highly 
probable  that  the  egg  membrane  keeps  out  bacteria  even  more 
effectually  than  does  the  shell.  Shell-less  eggs  like  those  of  the 
oviparous  snakes  are  well  protected  against  bacterial  infection 
by  the  thick  membrane,  even  though  the  eggs  are  deposited 
in  the  soil  and  in  decaying  rubbish.  It  has  also  been  suggested 
that  the  egg  membrane  contains  some  bacteriolytic  or  perhaps 
bactericidal  properties.  It  is  declared,  on  fairly  reliable  authority, 
that  fresh  egg  membrane  applied  to  buccal  inflammations  and 
threatened  abscesses  will  effect  a  prompt  cure.  The  Chinese 
have  used  egg  membranes  as  a  medicine  for  many  centuries. 


MEDICINAL   SUBSTANCES  1 97 

In  spite  of  shell  and  of  egg  membrane,  the  egg  is  gradually 
contaminated  more  and  more,  until  finally  complete  decomposi- 
tion has  taken  place.  Tests  made  by  European  investigators 
show  that  fresh  eggs  inoculated  with  various  molds  resist  penetra- 
tion completely  for  about  i  month.  After  8  weeks  species 
of  Cladosporum  had  entered.  In  12  weeks  Phytopthora  in- 
festans  developed  and  still  later  Rhizopus  nigricans.  Other  fungi 
which  finally  developed  in  the  interior  of  the  eggs  were  Clado- 
sporum herbarum,  Aspergillus  niger,  Penicillium  glaucum  and 
some  yeasts.  Fresh  egg  albumen  is  said  to  have  marked  bac- 
teriolytic properties,  but  such  properties  are  certainly  quickly 
lost  upon  exposure  to  the  air  and  light.  Some  eight  or  more 
species  and  varieties  of  bacteria  are  concerned  in  the  decomposi- 
tion of  eggs,  principally  aerobes.  Some  of  these  liberate  sul- 
phuretted hydrogen  {Bacillus  oogenes  hydro  sulphur  eus  group); 
others  belong  to  the  B.  coli  group  and  still  others  cause  decomposi- 
tion of  the  white  without  any  pronounced  color  development. 
Liquefaction  of  the  white  as  well  as  of  the  yolk  is  the  most  marked 
physical  change  in  eggs  undergoing  bacterial  decomposition. 
Mold  infection  is  very  generally  indicated  by  an  odor  of  mus- 
tiness.  Pronounced  mold  infection  is  further  indicated  by  spots 
shown  in  candling. 

19.  The  Bacteriological  Examination  of  Pharmaceutical  Preparations 

Thus  far  practically  nothing  has  been  done  as  to  the  bac- 
teriological examination  and  standardization  of  medicinal  sub- 
stances. There  is  a  popular  belief  that  the  ordinary  pharmaceu- 
ticals, particularly  the  tinctures  and  the  fluidextracts,  are  quite 
free  from  bacteria,  the  supposition  being  that  these  substances 
are  in  themselves  highly  antiseptic.  This  is  only  partially  in 
accord  with  facts.  Stronger  alcoholic  solutions  of  potent  drug 
constituents  no  doubt  inhibit  the  more  rapid  multiplication 
3f  most  bacteria  and  higher  fungi,  but  it  is  known  that  weak  solu- 


198  BACTERIOLOGICAL  METHODS 

tions   (i  per  cent.)  of  pilocarpine,  atropine,  cocaine,  morphine 
and  of  ergot,  on  standing  for  a  time,  show  many  millions  of  ■ 
bacteria  per  cc,  often  also  molds,  mold  spores  and  some  yeasts. ; 
The  variation  in  the  resisting  power  of  different  bacteria  toi 
different  medicinal  substances  is  noteworthy.     The  pus  staphy-j 
lococci  die  at  once  in  ether  and  in  a  saturated  solution  of  quinine, 
but  will  remain  active  in  a  10  per  cent,  solution  of  cocaine,  while  a  , 
2  per  cent,  solution  of  morphine  kills  them  in  24  hr.     The  same  j 
organisms  will  resist  the  action  of  pure  glycerin  for  6  to  8  days. 
Ten    per    cent,    iodoform,    glycerin,    camphorated    oil    (i-io), 
solutions   of    apomorphine    (0.2-20),    quinine    (i-io),    antipyrin 
(1-2),  and  cocaine  (i-io)  are  usually  quite  free  from  bacteria. 
The  coal-tar  derivatives  are  generally  considered   antiseptic  in 
property.     Aquae  are  frequently  found   to   contain  bacteria  in 
enormous  numbers   and  the   syrups  are  generally  more  or  less 
contaminated  with  yeasts,  bacteria   and   also   with   molds.     It 
is  known  that  weak  solutions  of  substances  intended  for  hy- 
podermic and  intravenous  use,  when  left  exposed  to  the  air  for  a 
time,    show    numerous    bacteria.     Tinctures    and    fluidextracts 
are  always  more  or  less  contaminated,  showing  organisms  in  direct 
proportion  to  age  and  the  degree  in  unsanitary  factory  conditions. 
Certain  medicinal  substances,  as  those  intended  for  hypodermic 
and  intravenous  use,  are  presumably  free  from  living  organisms. 
Undoubtedly  the  extensive  bacteriological  examination  of  medi- 
camenta  would  reveal  some  of  the  causes  which  are  responsible 
for  irregularities  in  drug  action,  and  would  explain  some  of  the 
hitherto  perplexing  phenomena  of  poisoning  resulting  from  the 
administration   of   certain   medicamenta   in   ordinary  medicinal 
doses. 

The   bacteriological    examination    of   medicamenta    may    be 
outlined  as  follows: 

Direct  microscopical  examination. 

1.  Bacteria. 

2.  Molds. 


MEDICINAL   SUBSTANCES  1 99 

3.  Mold  spores. 

4.  Yeasts. 
Plating  methods. 
Colon  bacillus  test. 
Tetanus  bacillus  test. 
Tests  for  the  staphylococcus  and  streptococcus  groups. 

In  securing  samples  for  examination,  the  precautions  necessary 
to  guard  against  outside  contamination  must  be  observed. 
Only  rarely  will  it  be  necessary  to  pack  the  samples  in  ice.  In 
the  preliminary  routine  of  the  laboratory,  many  of  the  more  ex- 
tensive contaminations  will  be  apparent  to  the  unaided  senses. 
Thus  a  change  in  color,  in  odor,  in  taste,  and  opacities  in  sub- 
tances  that  should  be  clear,  sediments  in  substances  that  should 
be  free  from  deposits,  etc.,  generally  indicate  decomposition 
changes  due  to  bacteria  and  other  organisms.  More  or  less 
cloudy  deposits  with  a  clear  supernatant  liquid  indicate  possible 
spore  sedimentation.  Extensive  contamination  by  bacteria, 
yeasts  and  molds  may  be  estimated  quantitatively  by  means 
of  the  hemacytometer  and  other  suitable  counting  devices. 
All  are  agreed  that  the  counts  for  medicinal  substances  intended 
for  administration  per  mouth  should  not  be  very  high.  How- 
ever, no  numerical  standards  have  as  yet  been  adopted.  It  is 
suggested  that  such  remedial  agents  are  unfit  for  use  when  they 
contain  bacteria  in  excess  of  5,000,000  per  cc.  and  yeasts  and  spores 
in  excess  of  500,000  per  cc.  Plating  methods  should  be  resorted 
to  in  order  to  ascertain  the  number  of  living  bacteria  present. 
The  potassium  tellurite  may  also  be  tried  in  order  to  ascertain 
microbic  invasion. 

All  substances  containing  gelatin,  intended  for  hypodermic 
use  or  for  application  to  mucous  membranes  or  to  abraded  skin 
surfaces,  should  be  tested  for  the  anaerobic  tetanus  bacillus 
{Bacillus  tetani).  Comparatively  large  quantities  should  be 
plated  in  large  Petri  dishes  or  in  Erlenmeyer  flasks  (using  agar 
media),  and  incubated  at  37°  C.  in  the  absence  of  oxygen.  Oxygen 
may  be  excluded  by  pouring  a  layer  of  sterile  olive  oil  over  the 


200  BACTERIOLOGICAL   METHODS 

medium  or  by  displacing  the  air  by  means  of  the  hydrogen  ap- 
paratus. The  colonies  which  appear  should  be  examined  micro- 
scopically to  ascertain  whether  or  not  the  characteristic  spore- 
forming  tetanus  bacillus  (drum  stick  bacillus,  the  Trommelschlager 
Bacillus  of  the  Germans)  is  present.  As  a  confirmatory  test,  a 
suspension  of  the  suspected  colony  should  be  injected  hypo- 
dermically  into  guinea-pigs  or  white  rats  and  symptoms  noted. 
Should  other  than  the  spore-forming  bacteria  be  present  in  the 
anaerobic  culture,  these  may  be  killed  by  pasteurizing  for  i  hr.  at 
80°  C,  at  which  temperature  all  organisms  excepting  the  spores 
of  the  tetanus  bacilli  are  killed.  Again  incubate  at  37°  C.  for 
several  days  and  examine  microscopically,  and  make  inocula- 
tion tests  as  already  suggested.  The  finding  of  a  single  tetanus 
bacillus  (as  represented  by  a  single  colony  in  the  anaerobic  culture) 
in  the  gelatin  renders  it  unfit  for  use. 

To  test  medicinal  substances  of  all  kinds  in  powdered  form, 
the  plating  method  must  be  relied  on  very  largely,  as  the  bacteria 
which  might  be  present  would  be  hidden  or  obscured  by  the 
granular  particles  present.  However,  extensive  yeast  and  mold 
contamination  could  be  detected  readily  and  estimated  quantita- 
tively by  the  direct  microscopical  method.  The  quahtative 
test  for  this  class  of  substances  is  very  largely  limited  to  the 
determination  of  the  absence  or  presence  of  the  colon  group, 
the  staphylococcus  group  and  the  streptococcus  group.  Face 
powders  and  dusting  powders  should  be  free  from  any  consid- 
erable contamination  with  the  pus-forming  organisms.  There 
should  be  uniformly  standard  methods  governing  the  manu- 
facture of  all  medicamenta,  intended  for  internal  or  external  use, 
which  must  be  touched  by  the  hands  of  the  manufacturer.  There 
must  be  absence  of  all  skin  diseases  and  of  transmissible  conta- 
gions of  all  kinds.  There  should  be  specific  requirements  as  to 
personal  cleanHness  and  the  sanitation  of  the  laboratory.  Just 
as  typhoid  carriers,  cholera  carriers  and  diphtheria  carriers  em- 
ployed as  servants  in  the  household  and  as  laborers  in  the  factory 


MEDICINAL   SUBSTANCES  20I 

may  spread  infections  among  those  with  whom  they  are  brought 
in  daily  contact,  so  may  the  manufacturing  pharmacist  convey 
disease  to  his  customers,  through  the  articles  which  he  offers 
for  the  cure  of  disease.  This  subject  should  receive  more  attention 
on  the  part  of  health  officials. 

Skin  and  scalp  infections  (acne,  boils,  abscesses,  carbuncles) 
are  traceable  to  the  use  of  powders  and  ointments.  The  more 
common  infections  which  may  be  carried  by  the  usual  hand  pre- 
pared face  powders  and  face  and  scalp  lotions  and  ointments  are 
pus  streptococci  and  staphylococci,  the  colon  bacillus  and  the 
tubercle  bacillus.  The  most  common  of  these  infections  are  the 
staphylococcus  group  of  pus  germs  and  the  germ  of  tuberculosis. 
Very  few  women  who  use  face  powders  persistently  for  a  long  time 
escape  without  more  or  less  severe  facial  infections.  Particularly 
is  this  true  of  women  who  use  the  more  or  less  irritating  chemical 
skin  renewers,  that  is,  so-called  cosmetics  which  act  by  removing 
the  superficial  epithelial  layers  of  the  skin.  The  use  of  these 
highly  irritating  agents  is  generally  followed  by  the  application 
of  the  germ-carrying  dusting  powders  and  ointments,  the  more  or 
less  raw  skin  favoring  the  infection. 

The  finger-nail  deposits  carry  many  different  kinds  of  germs 
accumulated  by  the  skin  and  scalp  scratching  process  and  through 
the  manifold  manipulations  of  all  manner  of  articles  during  the 
daily  work.  These  various  contaminations  may  be  transmitted 
to  the  hand  manufactured  toilet  and  face  preparations  offered 
for  sale  in  the  retail  drug  stores.  Among  the  bacteria  most 
commonly  found  with  the  finger-nail  deposits  are  streptococci, 
staphylococci  and  the  colon  bacilli.  Less  commonly  the  itch 
mite  and  the  larvae  of  intestinal  parasites  and  molds  are  found 
among  the  finger-nail  deposits. 

To  examine  finger-nail  deposits,  scrape  the  nail  of  the  thumb 
and  second  and  third  fingers  of  the  right  hand  (in  the  case  of 
right-handed  persons)  and  make  the  ordinary  smear  mounts, 
using  such  stains  and  reagents  as  may  be  required  to  bring  out 


202  BACTERIOLOGICAL  METHODS 

the  staining  properties  and  the  morphological  characteristics  of 
the  different  kinds  of  bacteria  and  of  other  organisms.  Cultural 
methods  may  be  desirable  for  purposes  of  identification. 

The  bacteriological  testing  of  ampuls  and  all  medicamenta  in- 
tended for  hypodermic,  intravenous  and  intramuscular  use  is 
reduced  to  great  simpHcity.  Since  these  substances  must  be 
absolutely  sterile,  the  finding  of  living  bacteria  (by  the  plating 
method)  would  be  proof  that  they  are  unfit  for  use.  It  must  be 
borne  in  mind,  however,  that  cloudiness  in  ampuls  (containing 
substances  which  should  be  clear)  does  not  necessarily  indicate 
bacterial  contamination,  as  this  condition  is  frequently  the  re- 
sult of  chemical  change,  possibly  occasioned  by  the  alkalinity 
of  the  glass  used  in  making  the  ampul's.  However,  all  ampuls 
which  show  cloudiness  when  they  should  be  entirely  clear  are 
to  be  rejected,  even  though  no  living  organisms  are  present. 
Oils,  salves  and  plasters  may  be  examined  directly  (microscopic- 
ally), noting  in  addition  to  bacteria  and  other  living  and  dead 
organisms,  possible  decomposition  changes  in  oils  and  fats,  as 
indicated  by  the  presence  of  the  characteristic  fat  crystals.  For 
plating,  oils  may  be  emulsified  with  measured  quantities  of  the 
liquefied  gelatin  or  agar  media  and  measured  quantities  poured 
into  Petri  dishes;  or  definite  quantities  (o.i  cc,  o.oi  cc,  o.ooi  cc, 
etc.)  may  be  planted  into  the  Petri  dishes  in  the  regulation  man- 
ner and  the  liquefied  gelatin  or  agar  poured  over  it  and  spread. 
Incubate  for  from  3  to  4  days  at  a  temperature  of  20°  C.  and 
count  the  colonies  formed.  Plasters  and  salves  may  be  plated 
by  liquefying  them  at  a  temperature  not  to  exceed  40°  C, 
making  the  desired  dilutions  with  sterilized  olive  oil. 

20.  The  Microscopical  and  Bacteriological  Examination  of  Syrups 

Syrups  are  very  important  products  extensively  used  in 
medical  and  pharmaceutical  practice  and  at  the  soda  fountain, 
and  for  practical  purposes  may  be  grouped  as  follows: 


SYRUPS  203 

I       I.  Medicinal  syrups. 

a.  Officinal,  simple  and  medicated. 

h.  Patent  and  proprietary  medicated  syrups. 

c.  Medicinal  preparations  containing  syrup  or  saccharine  substances. 

2.  Soda  fountain  syrups. 

3.  Fruit  juices  containing  sugar.     Fruit  juice  concentrates. 

4.  Syrups,  molasses,  treacle. 

Syrups  contain  cane  sugar  in  variable  amount.  Many  of 
them  also  contain  variable  amounts  of  invert  sugars.  The 
pharmaceutical  syrups  are  numerous  and  may  be  prepared  with 
sugar  or  simple  syrup.  The  chemicals  and  therapeutic  agents 
which  are  added  undoubtedly  have  some  influence  on  the  keeping 
quahties  of  the  preparations,  but  thus  far  no  one  has  made  any 
extensive  report  on  the  contaminations  of  medicinal  preparations 
containing  sugar  or  syrup.  The  soda  fountain  syrups  are  essen- 
tially sweetening  and  flavoring  agents  used  in  the  preparation 
of  the  familiar  soda  fountain  beverages,  ice  creams  and  sundaes, 
rhey  may  also  contain  physiologically  active  ingredients  such  as 
caffeine,  cocaine,  cocoa,  ginger,  etc. 

Manufacturers  have  more  or  less  difficulty  in  preparing  syrups 
tvhich  will  endure  without  spoiling.  Simple  syrup  and  the  fruit 
juices  in  particular  are  likely  to  undergo  yeast  fermentation,  and 
n  many  instances  there  is  also  mold  development,  more  com- 
nonly  the  Penicillium  glaucum.  The  contaminations  of  medicinal 
syrups  and  medicines  containing  syrup  are  very  variable.  Some 
)f  these  preparations  keep  for  a  long  time,  while  others  appear 
:o  be  quite  susceptible  to  the  invasion  of  yeasts.  In  many  in- 
stances the  initial  yeast  invasion  is  soon  followed  by  the  develop- 
nent  of  bacteria  and  also  mold. 

Among  the  organisms  which  are  most  likely  to  attack  syrups 
md  solutions  containing  sugar  are  the  so-called  potato  group  of 
Dacilli.  The  most  common  and  most  destructive  members  of 
:his  group  are  Bacillus  vulgatiis,B.  liodermos,  B.  mesentericus  fuscus , 
3.  mesentericus  ruber  and  B.  levaniformans,  which  latter  species 
s  really  the  group  type.     These  bacteria  are  widely  distributed  in 


204 


BACTERIOLOGICAL  METHODS 


nature,  occurring  in  the  soil  and  in  surface  waters,  and  because 
they  very  frequently  comtaminate  potato  cultures,  are  known  as 
the   potato   group.     They   are  spore  forming,  which  spores  are 


(50 


hi 


Ir   -^o 


cxx> 


w 


^s-A&jf  , 


Fig.  67, — Wild  and  pseudo-yeasts.  A,  S.  potnbe.  (After  Lindner);  B,  Torulce. 
{After  Pasteur);  C,  Mucor,  (i)  spores;  (2)  germinating  spores  and  mycelium;  D,  S. 
apiculatus;  E,  Mycoderma  vini. — (.4  iter  Bioletti.) 

remarkably  resistant  to  heat,  being  able  to  withstand  boihng  for 
2  hr.  Lafar  maintains  that  they  will  resist  the  temperature  of 
streaming  steam  for  6  to  7  hr.,  thus  making  them  the  most  re- 


SYRUPS  205 

sistant  of  all  bacterial  spores.  They  are  further  characterized  by 
their  ability  to  form  gummy  products  on  potato,  on  bread  and  in 
sugars. 

Bacillus  mesentericus  niger  and  B.  granulatus  mesentericus 
are  also  members  of  the  potato  group  and  may  be  found  in  sugars 
and  syrups.  Leuconostoc  mesenteroides  and  Bacterium  gelatinosum 
betcB,  which  are  frequently  found  in  sugar  beet  juice,  are  evidently 
related  to  the  potato  group.  Bacillus  gummosus,  also  a  gum 
former,  is  frequently  found  in  digitalis  infusions.  This  latter 
bacillus  is  comparatively  large,  feebly  motile,  forms  spores  and 
produces  lactic  and  butyric  acids.  Another  organism  which  is 
probably  closely  related  to  the  potato  group  is  the  Bacterium 
mesentericus  panis  viscosum,  the  cause  of  stringy  or  slimy  bread. 
All  of  the  organisms  named  are  Gram  positive,  are  spore  formers, 
liquefy  gelatin  and  are  motile,  having  ilagellae.  The  principal 
biologic  characteristics  of  the  group  may  be  given  as  follows: 

1.  They  form  gums  (levan)  from  sugars. 

2.  The  spores  are  very  resistant  to  heat. 

3.  They  have  a  very  low  nutrient  requirement. 

Very  minute  quantities  of  sugar  are  sufficient  to  induce  them 
to  grow  and  multiply,  resulting  in  the  transformation  of  some  of 
the  sugar  into  a  gum  known  as  levan. 

From  the  above  general  characteristics  of  the  sugar  bacteria 
it  is  almost  self-evident  how  they  may  cause  very  serious  harm  to 
solutions  of  all  kinds  containing  sugar.  The  highly  resistant 
spores  make  thorough  sterilization  difficult  and,  since  high  tem- 
peratures cause  inversion  of  sugars,  the  use  of  the  autoclave  and 
repeated  and  prolonged  heating  at  the  boiling  temperature  are 
frequently  not  permissible.  In  cases  where  the  use  of  the  autoclave 
is  permissible,  a  single  exposure  at  a  temperature  of  120°  C. 
for  a  period  of  30  min.  is  sufficient  to  kill  the  spores.  More 
generally  the  fractional  method  of  heat  sterilization  must  be 
employed. 

It  is  a  noteworthy  fact  that  after  the  gum   formers  have 


2o6 


BACTERIOLOGICAL  METHODS 


once  gained  access  to  the  syrup  they  are  not  easily  exterminated. 
This  emphasizes  the  importance  of  great  care  and  cleanliness  in 
the  preparation  of  all  syrups.  It  is  also  a  fact  that  high  con- 
centrates of  sugar  are  not  so  liable  to  be  attacked  as  are  the 
weaker  solutions.     The  most  favorable  strength  of  sugar  solution 


'.^c^. 


# 


h 


(^ 


Fig.  68. — Bacillus  californiensis  isolated  from  the  roots  of  the  sugar  beet.  A 
typical  gum  former  found  in  the  soil,  on  the  roots  and  in  the  surface  tissues  of  the 
sugar  beet  and  in  the  juice  of  the  sugar  beet,  a,  Beet  root  cells  showing  a  mixture 
of  cell  plasm  and  B.  californiensis  (mycoplasm);  b,  epidermal  cells  of  the  root  show- 
ing bacteria  within  the  cell  and  also  on  the  exterior;  c,  a  bit  of  mycoplasm  removed 
from  the  cell  by  pressure;  d,  B.  californiensis  removed  from  the  cells  by  pressure; 
e,  B.  californiensis  from  a  pure  culture  in  beet  root  gelatin;  /,  zoogloea  form  of 
B.  cal.;  g,  gelatinized  form  of  B.  cal.;  i,  a  single  chain  highly  magnified  (X  450 
to  1000). 


is  about  20  per  cent.  Development  may,  however,  take  place  in 
60  per  cent,  solutions.  The  organisms  are  facultatively  anaerobic 
and  their  growth  is  greatly  accentuated  by  free  aeration.  Com- 
pletely filling  the  containers  will  materially  check  and  even 
completely  prevent  the  growth  of  the  bacteria. 


SYRUPS  207 

The  bacterial  diseases  of  syrups  and  substances  containing 
sugar  are  by  far  the  most  important  and  the  most  difiicult  to 
combat.  We  must,  however,  not  forget  the  far  more  common 
fermentation  and  decomposition  changes  induced  by  the  yeasts 
and  molds.  Yeasts  are  very  likely  to  attack  the  openly  exposed 
fruit  juices  and  fruit  syrups  at  the  soda  fountain.  Crushed 
fruits  of  the  soda  fountain  may  contain  yeasts  and  also  bacteria 
and  molds.  Fruit  products  invaded  by  yeasts  are  no  longer 
suitable  for  use  at  the  soda  fountain.  The  attempt  to  render 
fermenting  fruit  juices  usable  by  heating  is  not  practicable  as  the 
natural  flavor  is  lost,  and  the  attempt  to  use  such  fruits  or  fruit 
juices  would  prove  disastrous  to  the  soda  fountain  business. 

Little  is  known  regarding  the  influence  of  the  therapeutically 
active  ingredients  of  the  medicinal  syrups  on  the  development  of 
the  sugar-destroying  bacteria  but  it  would  appear  from  the  reports 
of  some  observers  that  they  do  not  materially    check    them. 
It  is  quite  evident  that  the  carbonated  sugar-bearing  soda  foun- 
tain drinks  are  just  as  liable  to  bacterial  invasion  as  are  the 
noncarbonated  soft  drinks.      The  sugar-destroying  gum  formers 
frequently  do  great  damage  to  the  soda  bottling  business,  at  times 
j:  ruining  the  entire  output.     The  trouble  may  be  slow  in  making 
|!itself  evident.     For  a  long  time  all  appears  to  be  well  at  the 
ll  factory  but  gradually  complaints  come  in  from  the  dealers  (dis- 
tributors) and  the  consumers.     It  is  claimed  that  ''gelatinous 
lumps"  appear  in  the  drink  and  that  the  taste  is  insipid  and  not 
I  sufficiently  sweet.     Very  naturally  these   complaints   are   detri- 
I mental  to  the  business.     With  proper  attention  to  details  at  the 
5  factory  the  trouble  could  have  been  avoided.     In   the  manu- 
jfacture  of  soda  fountain  syrups  and  medicinal  syrups,  one  of  the 
most  important  essentials  is  thorough  sterilization  of  the  syrup 
to  be  used.     The  other  ingredients  which  enter  into  their  com- 
I  position   should   also   be   thoroughly   sterilized.     All   containers 
should  be  thoroughly  sterilized  and  they  should  be  completely 


208 


BACTERIOLOGICAL   METHODS 


filled  while  the  containers  and  the  syrup  are  still  hot  and  then  her- 
metically sealed  by  means  of  sterilized  stoppers. 

In  the  case  of  medicinal  syrups  and  sugar-bearing  medicines 
which  are  contaminated  by  bacteria,  it  must  be  borne  in  mind 
that  the  active  constituents  present  are  also  more  or  less  com- 
pletely decomposed.  Such  substances  should  be  quite  free  from 
contamination  and  the  presence  of  marked  contamination  should 


Fig.  69. — Stab  culture  appearance  of  B.  californiensis  in  beef  extract  gelatin 
tubes.  I,  Appearance  of  growth  on  third  day  after  inoculation;  2,  deep  stab  cul- 
ture 24  hr.  old,  from  tube  (i);  3,  same  as  (2)  36  hr.  old.  Liquefaction  of  gela- 
tin is  noticeable;  4,  same  as  (2)  3  days  old.  In  the  course  of  2  weeks  the  entire 
contents  of  tube  became  liquefied. 


form  the  basis  for  the  condemnation  of  such  products.  The 
adoption  of  a  numerical  bacterial,  yeast  and  mold  standard  would 
appear  highly  desirable. 

Among  the  products  which  are  classed  with  the  syrups  are 
syrup  or  molasses  and  treacle  from  the  sugar  cane,  and  the  sorghum 
molasses  of  the  central  and  northern  states,  maple  syrup  and 
other  syrups  of  commerce  including  the  so-called  corn  syrup  which 


SYRUPS 


209 


IS  largely  starch  glucose,  the  glucose  syrups,  honey  and  other 
syrupy  substances  used  as  a  food  and  condiment.  As  a  rule 
these  do  not  come  to  the  notice  of  the  bacteriologist.  A  micro- 
scopical examination  may  be  made  occasionally.  For  example, 
the  finding  of  pollen  grains  may  be  the  means  of  distinguishing 
between  true  and  imitation  honey.  Molds  occasionally  attack 
syrups  and  less  frequently  yeast  cells  may  be  found  in  some  of  the 
improperly  stored  syrups. 


Fig.  70. — Streak  culture  appearance  of  B.  californiensis  on  beef  extract  gelatin,  i, 
24  hr.  old;  2,  48  hr.  old;  3,  72  hr.  old;  4,  4  days  old. 


The  bacteriological  examination  of  sugars,  candies  and  condi- 
ments is  wholly  incidental  and  need  not  be  discussed.  Analysis 
Df  this  class  of  substances  is  left  almost  entirely  to  the  chemist 
md  the  micro-analyst. 

The  bacteriological  and  microscopical  examination  of  crushed 
fruits,  fruit  juices  and  fruit  juice  concentrates  is  much  the  same 
is  for  syrups.  The  crushed  fruits  at  the  soda  fountain  are  very 
prone  to  yeasty  fermentation  during  the  hot   summer  months. 


2IO  BACTERIOLOGICAL  METHODS 

Grape  juice  is  apt  to  become  moldy.  The  nature  of  the  con- 
tamination will  depend  upon  the  relative  amounts  of  sugar  and 
acids  present. 

• 

21.  The  Microscopical  and  Bacteriological  Examination  of  Fer- 
mented Foods  and  Drinks 

The  examination  of  alcoholic  drinks  and  other  fermented 
liquids  and  fermented  food  substances  including  certain  fermented 
products  used  in  the  preparation  of  foods,  on  the  part  of  the 
bacteriologist,  is  of  minor  importance  and  is  largely  supplementary 
to  the  analyses  of  the  chemist  and  the  organoleptic  testings  of 
the  expert  taster.  The  methods  of  procedure  will  be  largely 
limited  to  the  microscopical  examination  of  concentrates,  of  natural 
and  centrifugalized  sediments,  of  sedimentary  suspensions  and 
of  surface  formations  or  deposits,  with  a  view  to  the  detection  of 
added  or  other  impurities  and  the  recognition  of  abnormal  fer- 
mentative changes,  and  invasions  by  objectionable  bacteria,  yeasts 
and  molds.  *' Diseased"  or  ''sick"  wines,  beers,  porters,  ales, 
vinegars,  pickles,  sauerkraut,  etc.,  should  be  carefully  examined 
as  to  the  quantity  and  identity  of  the  objectionable  organisms 
present.  In  order  that  the  report  of  the  bacteriologist  may 
supplement  the  report  of  the  expert  taster,  it  is  absolutely  essential 
that  the  bacteriologist  have  a  thorough  knowledge  of  the  micro- 
scopical appearance  of  normally  fermented  products.  This 
knowledge  may  be  gained  only  through  experience.  The  yeasts 
and  other  organisms  concerned  in  normal  wine  fermentation  are 
wxll  known  to  the  specialists  who  have  made  a  long  study  of  wine 
ferments. 

There  appears  to  be  no  recognized  standard  as  to  the  number 
or  kind  of  organisms  which  may  be  permissible  in  properly  fer- 
mented and  properly  clarified  wines  and  in  other  fermented 
drinks,  nor  does  the  present  status  of  the  subject  warrant  the 
adoption  of  numerical  limits  as  to  the  organisms  present.     There 


FERMENTED   FOODS   AND   DRINKS 


211 


are,  however,  many  instances  in  which  the  findings  of  the  bacteri- 
Dlogist  may  be  final  and  conclusive  as  to  the  quality  and  purity 
3f  the  wine  or  other  fermented  alcoholic  beverages  or  of  fermented 
[ood  products.     If,  for  example,  there  is  abundant  mold  forma- 


FiG.  71. — Development  of  Miicor  mucedo.  a,  b,  c,  d,  stages  in  the  formation  of 
he  zygospore;  d,  mature  zygospore;  e,  f,  endospore  formation;  g,  endospores;  h, 
erminating  spore,  the  beginning  of  the  new  zygospore  forming  cycle.  The  appear- 
nce  of  the  stalks  and  the  spore  bearing  capsules  explains  why  this  is  called  the 
pin  cushion  fungus."  Related  molds  occur  on  stale  bread,  on  fruits,  on  damp 
loves  and  leather  generally.  It  is  the  cause  of  a  fatal  infectious  disease  in  house- 
ies. 


ion  in  a  product  which  normally  should  be  free  from  such  organ- 
sms,  then  the  product  should  be  pronounced  unfit  for  human  use. 
^gain  it  may  be  possible  to  recognize  abnormal  bacterial  or 
)erhaps  abnormal  yeast  development  as  the  causes  of  the  deterio- 


15 


212  BACTERIOLOGICAL  METHODS 

ration  and  undoubtedly  the  microscope  alone  will  in  most  in- 
stances reveal  the  presence  of  numerous  abnormal  and  objec- 
tionable organisms,  even  before  the  expert  taster  has  been  able 
to  appreciate  any  abnormal  alteration  in  flavor  or  in  bouquet. 

The  following  is  a  very  brief  outline  of  the  principal  fermen- 
tations concerned  in  the  manufacture  of  alcoholic  beverages. 

A.  Alcoholic  Fermentation. — The  alcohol  forming  ferments  or 
zymases,  or  yeast  ferments  proper,  are  by  far  the  most  common 
and  most  widely  distributed  in  nature  and  the  most  important 
from  a  commercial  and  economic  standpoint.  The  zymases  act 
upon  sugars  splitting  these  into  alcohol  and  carbonic  acid  gas, 
thus  acting  upon  the  end  products  formed  by  the  diastases  and 
preparing  them  for  the  action  of  the  acid  forming  ferments. 

Zymases  are  formed  by  a  great  variety  of  plants  and  animals, 
more  generally  by  the  so-called  yeast  plants  (the  Saccharomyces 
and  Torula  groups).  The  alcohol-generating  enzymes  formed 
by  these  plants  are  capable  of  being  isolated  or  separated  from 
the  living  cells  which  form  them  and  may  continue  the  fermenta- 
tive activities  indefinitely.  Alcoholic  fermentation  is  by  no 
means  a  simple  process.  The  degree  of  alcohol  production 
and  by-product  formation  varies  greatly,  depending  upon  a  great 
variety  of  factors  and  influences.  To  enter  into  a  fuller  dis- 
cussion of  the  details  of  the  fermentative  processes  and  a  de- 
scription of  the  organisms  involved,  is  not  practicable  or  essential 
for  the  present  purpose.  The  number  of  saccharine  substances 
capable  of  undergoing  alcoholic  fermentation  is  legion,  and  it 
has  thus  far  not  been  possible  to  ascertain  the  number  and  variety 
of  yeast  organisms  and  associated  organisms  which  are  involved 
in  the  multitudinous  fermentations  (natural  and  artificial) 
resulting  in  the  formation  of  alcohol.  In  commercial  practice: 
(in  the  manufacture  of  wine,  beer,  brandy,  etc.),  a  distinction  is 
made  between  upper  yeasts,  lower  yeasts,  wild  yeasts,  etc.  In 
some  breweries  lower  yeasts  are  the  chief  fermenters  used  and  in 
others  the  upper  yeasts  are  preferred.     For  example,  it  is  claimed 


FERMENTED   POODS   AND   DRINKS  213 

that  the  use  of  bottom  yeast  (Unterhefe)  makes  it  easier  to  guard 
against  the  entrance  of  wild  yeasts  and  other  objectionable 
organisms.  On  the  other  hand  it  is  claimed  that  the  use  of  the 
upper  yeast  (Oberhefe)  yields  a  better  quality  of  beverage.  These 
ire  factors  of  the  greatest  importance  to  the  bacteriologists  and 
jymologists  employed  by  the  breweries  but  concern  the  food 
Dacteriologist  but  little. 

The  following  are  some  of  the  more  important  yeast  organisms 
:oncerned  in  alcohohc  fermentation,  giving  the  principal  fermenta- 
:ive  activities  of  each.  Hansen's  differentiation  between  the 
genera  Saccharomyces  and  Torula  is  based  upon  sporulation. 
jaccharomyces  forms  spores  (Ascospores;  usually  four  spores  in 
^ach  ascus  or  spore  sac,  rarely  eight)  whereas  Torula  does  not 
orm  spores.  According  to  some  authorities  this  is  not  a  practical 
)asis  of  differentiation. 

accharomyces 

cerevisea  Hansen.     A  typical  top  yeast. 

pastorianus,  Hansen.     A  bottom  yeast. 

intermedins,  Hansen.     A  rather  feebly  acting  top  yeast. 

validus,  Hansen.     A  top  yeast. 

ellipsoideus,  Hansen.     A  typical  bottom  yeast. 

turhidans,  Hansen.     A  bottom  yeast  the  cause  of  turbidity. 

willianus,  Saccardo.     A  flavor-producing  yeast. 

boyanus,  Saccardo.     Causes  turpidity  in  beer  and  wine. 

logos,  van  Laer.     A  bottom  yeast  developing  a  flavor.  '    ' 

thermanitonum,  Johnson.     A  rapidly  acting  ferment. 

ilicis,  Gronlund.     A  bottom  yeast;  isolated  from  Ilex. 

aquifolii,  Gronlund.     Also  isolated  from  Ilex  species. 

pyriformis,  Ward.     Found  in  ginger  beer. 

vordermanni,  W.  and  P.  Isolated  from  Arrak. 

sake,  Yabe.     Active  in  the  fermentation  of  sak6. 

batata,  Saito.     In  yam  brandy. 

cartilaginosus,  Lindner.     Isolated  from  Kephir. 

multisporus,  Hansen.     A  top  yeast. 

mali,  Kayser.     A  cider  ferment. 

marxianus,  Hansen.     A  wine  ferment. 

exiguus,  Hansen.     In  beer  wort. 

jorgensenii,  Lasche.     Causes  turbidity. 

zopfii,  Artari.     Found  in  syrup. 


214  BACTERIOLOGICAL  METHODS 

bailii,  Lindner.     In  beerwort. 

hyalosporus,  Lindner.     In  beerwort. 

rouxi,  Butroux.     Found  in  fruit  juices. 

soya,  Saito.     In  soya  sauce. 

unisporus,  Hansen.     In  dutch  cream. 

flava  lacHs,  Krueger.     Found  in  cheesy  butter. 

hanseni,  Zopf.     In  cotton  seed  meal. 

minor,  Engelman.     Found  in  bread. 

membranaefaciens,  Hansen. 

anomalans,  Hansen.     Causing  a  fruity  flavor. 

saturnus,  Klocker.     Isolated  from  soil. 

acidi  lactici,  Grotenfeldt.     A  milk-curdling  yeast. 

fragilis,  Jorgensen.     Found  in  Kephir. 

barkeri,  Saccardo.     In  ginger  beer. 

ludwigii,  Hansen.     From  oak  bark  extract. 

comesii,  Covara.     From  millet  seed. 

octosporus,  Bevjerinck.     On  dried  currants. 

mellacei,  Jorgensen.     A  top  yeast  developing  a  pleasant  odor. 

guttulatus,  Robin.     Found  in  a  rabbit. 

capsularis,  Schionning.     From  soil. 

According  to  Hansen,  Torulas  also  occur  in  great  variety. 
The  Levure  de  sel  is  a  yeast  capable  of  developing  in  a  lo  to  15  per 
cent,  sodium  chloride  solution.  Those  desiring  to  obtain  detailed 
information  regarding  the  complete  fermentation  processes  in- 
volved in  the  brewing  of  beer  and  other  fermented  drinks,  must 
consult  the  special  technical  treatises  of  which  there  are  many 
available. 

B.  Acid-forming  Ferments. — Dilute  alcohol  upon  standing 
exposed  to  the  air,  gradually  becomes  sour,  losing  its  alcohol 
more  and  more.  This  loss  of  alcohol  and  gain  in  acidity  is  due 
to  the  action  of  ferments  which  split  the  alcohol  into  acetic  acid 
and  water.  The  organisms  which  produce  the  acid-forming  fer- 
ments or  enzymes  mostly  belong  to  the  group  bacteria  (bacilli). 
The  more  common  and  important  species  are  Mycoderma  {Bacillus) 
aceti,  B.  Pasteurianum,  B.  kutzingianum,  B.  oxydans  and  B. 
acetosum.  The  yeast  Saccharomyces  mycoderma  is  also  capable  ofj 
forming  acetic  acid.  The  vinegar  organisms  are  most  active! 
at  a  temperature  of  25^  C.  to  30°  C.     They  are  very  slowly  active] 


FERMENTED   FOODS   AND   DRINKS 


215 


at  io°  C.  and  are  killed  at  a  temperature  above  35°  C.  The 
so-called  mother  of  vinegar  consists  of  an  agglutinated  mass  of 
Mycoderma  aceti  and  is  used  as  a  starter  in  the  manufacture  of 
vinegar.  Thus  far  it  has  not  been  possible  to  isolate  the  vinegar 
ferment  or  enzyme  from  the  living  cells  which  form  it. 

There  are  also  acids  of  nonalcoholic  origin  formed  by  living 
ferments,  such  as  oxalic  acid,  malic  acid,  citric  acid  and  others, 


Fig.  72, — Types  of  yeast  organisms  and  yeast  sporulation.  A,  Saccharomyces 
pasteurianus  showing  spore  formation  in  fours  and  eights  {after  Bioletti);  B, 
Schizosaccharomyces  octosporus,  showing  simple  septation  instead  of  budding,  and 
spore  formation  (after  Schionning);  C,  Saccharomyces  anomalus,  vegetative  cells 
and  spore  sacs. — {Marshall,  after  Kayser.) 


which  appear  to  be  derived  from  the  direct  fermentation  of 
sugars.  Citric  acid  is  forriied  from  sugars  through  the  activity 
of  two  fungi,  Citromyces  pfefferianus  and  C.  glaher.  Saccharo- 
myces hansenii  forms  oxalic  acid  from  mannit  and  galactose,  with- 
out the  intermediary  alcohol  formation. 

The  following  are   the  more  important  products  in  which 
there  is  alcohol  formation  through  the  action  of  yeast  organisms. 


2l6 


BACTERIOLOGICAL  METHODS 


I.  Whiskey  and  Brandy. — Whiskey  and  brandy  are  alcoholic 
beverages  with  an  alcohol  content  ranging  from  about  44  per 
cent,  to  55  per  cent,  (by  volume).  Whiskey  (Spiritus  frumenti 
of  the^^U.  S.  P.  and  Schnapps  of  the  Germans)  is  usually  made 
from  grain  as  rye,  wheat,  barley  and  corn.     Brandy  (Branntwein) 


I  €'^ 


^ 


® 


/f 


Fig.  73. — Wine  and  beer  yeasts.  A,  Saccharomyces  ellipsoides  showing  the 
young  and  vigorous  cells;  B,  the  same  cells  old  (i)  and  dead  (2);  C,  S.  cerevisea  as 
top  yeast  and  D,  S.  cerevisece  as  bottom  yeast. — {Marshall.) 

is  usually  made  from  grapes.  In  the  manufacture  of  both  whiskey 
and  brandy  there  is  alcoholic  fermentation  followed  by  distilla- 
tion, with  or  without  the  addition  of  coloring  substances,  as 
caramel.  In  bothjwhiskey  and  brandy,  certain  collateral  prod- 
ucts of  distillation^known  as  congeners,  such^as  flavor  (bouquet), 
aldehydes,  ethers,  trace  of  fusel  oil,  trace  of  fruit  or  grain  color,  of 


WHISKEY   AND   BRANDY  21 7 

acids,  etc.,  are  present.  These  congeners  are  normally  present 
and  vary  somewhat,  dependent  upon  variations  in  the  method 
of  distillation,  slight  differences  in  the  quality  of  the  grain  or 
fruit  used,  the  process  of  fermentation,  temperature,  etc.  With 
ageing  whiskey  as  well  as  brandy  undergo  complex  changes  (chem- 
ical as  well  as  fermentative)  indicated  by  changes  in  color,  odor 
and  taste.  These  slow  changes  appear  to  be  largely  zymotic  in 
nature  but  they  are  not  well  understood. 

Whiskey  and  brandy  may  be  made  from  all  substances  ca- 
pable of  undergoing  alcoholic  fermentation,  such  as  rice,  wheat, 
barley,  rye,  oats,  potatoes,  apples,  pears,  berries  of  all  kinds,  etc. 
Any  apparatus  so  constructed  and  equipped  as  to  vaporize  and 
carry  over  and  condense  the  alcohol  existing  in  the  fermented 
product,  may  be  used  in  distillation.  In  the  process  of  distilla- 
tion certain  congeners  are  always  carried  over  with  the  alcohol 
and  these  constitute  normal  inclusions  of  the  brandy  or  whiskey. 
If  the  congeners  are  poisonous  or  otherwise  objectionable,  then 
the  distillate  containing  them  is  also  poisonous  or  otherwise 
objectionable  and  may  render  the  product  unsuitable  for  human 
use.  These  poisonous  congeners  evidently  exist  in  certain 
products  of  alcoholic  distillation  and  should  be  more  carefully 
investigated. 

Alcohol  per  se  (less  all  congeners)  is  a  protoplasmic  poison. 
Small  quantities,  when  taken  into  the  system  are  oxidized  and 
in  so  far  as  it  is  oxidized,  alcohol  is  a  food,  but  because  of  its 
toxic  character,  alcohol  can  never  be  used  as  a  food  having 
practical  value  as  such. 

Rectified  whiskey  or  brandy  is  redistilled  or  double  distilled 
whiskey  or  brandy.  As  a  result  of  this  redistillation  there  is  an 
increase  in  the  alcoholic  strength,  with  a  decrease  in  the  amount 
of  fusel  oil,  a  change  or  decrease  in  the  congeners,  a  change  in  the 
color  and  bouquet  or  flavor,  etc.  As  generally  comprehended 
rectification  implies  purification  and  increase  in  alcoholic  strength, 
without  foreign  additions  of  any  kind.     Adding  coloring  sub- 


2l6 


BACTERIOLOGICAL  METHODS 


I.  Whiskey  and  Brandy. — Whiskey  and  brandy  are  alcoholic 
beverages  with  an  alcohol  content  ranging  from  about  44  per 
cent,  to  55  per  cent,  (by  volume).  Whiskey  {Spiritus  frumenti 
of  the^U.  S.  P.  and  Schnapps  of  the  Germans)  is  usually  made 
from  grain  as  rye,  wheat,  barley  and  corn.     Brandy  (Branntwein) 


^      ^^    I  ^'^ 


® 


jf 


Fig.  73. — Wine  and  beer  yeasts.  A,  Saccharomyces  ellipsoides  showing  the 
young  and  vigorous  cells;  B,  the  same  cells  old  (i)  and  dead  (2);  C,  S.  cerevisecB  as 
top  yeast  and  D,  S.  cerevisea  as  bottom  yeast. — {Marshall.) 

is  usually  made  from  grapes.  In  the  manufacture  of  both  whiskey 
and  brandy  there  is  alcoholic  fermentation  followed  by  distilla- 
tion, with  or  without  the  addition  of  coloring  substances,  as 
caramel.  In  bothfwhiskey  and  brandy,  certain  collateral  prod- 
ucts of  distillation^known  as  congeners,  such*as  flavor  (bouquet), 
aldehydes,  ethers,  trace  of  fusel  oil,  trace  of  fruit  or  grain  color,  of 


WHISKEY  AND  BRANDY  21 7 

acids,  etc.,  are  present.  These  congeners  are  normally  present 
and  vary  somewhat,  dependent  upon  variations  in  the  method 
of  distillation,  slight  differences  in  the  quality  of  the  grain  or 
fruit  used,  the  process  of  fermentation,  temperature,  etc.  With 
ageing  whiskey  as  well  as  brandy  undergo  complex  changes  (chem- 
ical as  well  as  fermentative)  indicated  by  changes  in  color,  odor 
and  taste.  These  slow  changes  appear  to  be  largely  zymotic  in 
nature  but  they  are  not  well  understood. 

Whiskey  and  brandy  may  be  made  from  all  substances  ca- 
pable of  undergoing  alcoholic  fermentation,  such  as  rice,  wheat, 
barley,  rye,  oats,  potatoes,  apples,  pears,  berries  of  all  kinds,  etc. 
Any  apparatus  so  constructed  and  equipped  as  to  vaporize  and 
carry  over  and  condense  the  alcohol  existing  in  the  fermented 
product,  may  be  used  in  distillation.  In  the  process  of  distilla- 
tion certain  congeners  are  always  carried  over  with  the  alcohol 
and  these  constitute  normal  inclusions  of  the  brandy  or  whiskey. 
If  the  congeners  are  poisonous  or  otherwise  objectionable,  then 
the  distillate  containing  them  is  also  poisonous  or  otherwise 
objectionable  and  may  render  the  product  unsuitable  for  human 
use.  These  poisonous  congeners  evidently  exist  in  certain 
products  of  alcoholic  distillation  and  should  be  more  carefully 
investigated. 

Alcohol  per  se  (less  all  congeners)  is  a  protoplasmic  poison. 
Small  quantities,  when  taken  into  the  system  are  oxidized  and 
in  so  far  as  it  is  oxidized,  alcohol  is  a  food,  but  because  of  its 
toxic  character,  alcohol  can  never  be  used  as  a  food  having 
practical  value  as  such. 

Rectified  whiskey  or  brandy  is  redistilled  or  double  distilled 
whiskey  or  brandy.  As  a  result  of  this  redistillation  there  is  an 
increase  in  the  alcoholic  strength,  with  a  decrease  in  the  amount 
of  fusel  oil,  a  change  or  decrease  in  the  congeners,  a  change  in  the 
color  and  bouquet  or  flavor,  etc.  As  generally  comprehended 
rectification  implies  purification  and  increase  in  alcoholic  strength, 
without  foreign  additions  of  any  kind.     Adding  coloring  sub- 


2l8  BACTERIOLOGICAL  METHODS 

stances  or  flavoring  agents  to  raw  (unaged)  whiskey  or  brandy  so 
as  to  imitate  the  product  which  has  been  allowed  to  age  natu- 
rally, constitutes  adulteration  under  the  Federal  Pure  Food  and 
Drugs  Act.  Adding  whiskey  or  brandy  to  alcohol  (ethyl)  com- 
monly known  as  rectified  spirits,  does  not  make  rectified  whiskey 
or  brandy. 

Various  medicamenta  may  be  added  to  whiskey  and  brandy, 
such  as  caraway,  aloes,  juniper  berries,  absinthium,  etc.  Many 
alcoholic  beverages  are  sold  to  the  unsuspecting  public  under  the 
guise  of  tonics  and  blood  purifiers. 

2.  Beer. — Beer  is  a  fermented  drink  generally  made  from 
barley.  The  carefully  selected  grain  is  washed  in  running  water 
and  then  macerated  in  water  to  induce  germination.  This 
process  liberates  the  ferment  diastase  which  occurs  in  the 
grain  and  this  enzyme  acts  upon  the  starch  present  converting  it 
into  saccharine  compounds.  The  saccharine  compounds  are 
next  acted  on  by  the  yeasts  {Saccharomyces  cerevisece  and  other 
species)  which  convert  the  sugars  into  alcohol.  Hops  are  added 
to  give  the  beer  a  bitter  taste  and  also  for  the  purpose  of  in- 
fluencing the  fermentation  process  favorably.  After  the  al- 
cohoKc  fermentation  is  completed,  the  product  is  filtered,  clarified, 
pasteurized  and  occasionally  preserved  by  adding  salicylic  acid 
or  other  preservative.  The  alcoholic  content  of  beer  varies  from 
about  1.50  to  6  per  cent.  Some  beers  are  fortified  by  adding 
alcohol.  There  are  many  kinds  or  brands  of  beer,  differing  in 
color,  flavor,  taste  and  consistency. 

Brewers  must  observe  great  caution  to  guard  against  the  inva- 
sion of  objectionable  organisms  as  bacteria,  yeasts  and  mold, 
which  might  vitiate  the  normal  or  desirable  process  of  fermenta- 
tion. In  spite  of  all  precautions,  things  often  go  wrong.  The 
wort  may  undergo  sour  or  other  objectionable  fermentation  and 
as  a  result  the  entire  lot  may  have  to  be  rejected.  Wild  yeasts 
may  gain  the  upper  hand  and  ruin  the  beer.  Even  after  the 
product  is  finished  and  placed  in  the  containers,  abnormal  fer- 


BEER 


219 


mentations  may  be  set  up  by  various  bacteria,  yeasts  and  mold, 
causing  more  or  less  serious  spoiling  and  even  complete  deteriora- 
tion. The  following  are  the  more  common  beer  diseases  which 
may  be  brought  to  the  attention  of  the  food  bacteriologist. 

a.  Ropiness. — This  is  quite  common.  The  beer  becomes  thick 
and  mucilaginous  capable  of  being  drawn  out  into  threads.  Two 
species  of  bacteria  cause  ropy  beer;  Bacillus  viscosus  I  and  B. 


Fig.  74. — Saccharomyces   cerevisecE.     The   variety   known   as  brewers' 

yeast. — {Oberhefe.) 


top 


viscosus  11.  These  bacteria  are  rod-shaped  and  measure  0.8  by 
1.6-2.4  microns.  Bacillus  I  gives  rise  to  yellowish-white  viscous 
patches  on  the  surface  of  the  beer  whereas  bacillus  II  does  not 
develop  such  patches.  B.  viscosus  III  has  been  isolated  from 
British  ropy  beer.  The  ropiness  results  from  a  change  in  the  cell- 
wall  of  the  bacterium  and  not  from  any  chemical  change  in  the 
beer  itself.     Ropiness  may  also  be  caused  by  a  mold,  Dematium 


220 


BACTERIOLOGICAL  METHODS 


pullulans,  which  shows  septate  branching  hyphal  filaments  and 
yeast-like  sporulation,  which  might  be  mistaken  for  yeast  cells. 
b.  Turning  or  Souring  of  Beer. — Soured,    turned  or  spoiled 
beers  have  a  disagreeable  taste  and  odor  and  are  no  longer  clear  or 
brilliant   and   sedimentary   deposits   are   usually   found.     Beers 


Fig.  75- — Saccharomyces  cerevisecs.  The  variety  known  as  brewers'  bottom 
yeast  (Unterhefe).  a,  Spore  formation;  b,  elongated  cells  (rudimentary  filaments 
or  hyphae). 


containing  only  a  small  amount  of  hops  or  of  hop  extract  and 
which  are  low  in  alcohol  and  inadequately  filtered,  pasteurized 
and  improperly  bottled,  are  likely  to  spoil.  The  most  common 
cause  of  this  kind  of  spoiling  is  due  to  the  aerobic  acetic  acid 
bacteria  which  are  particularly  apt  to  do  great  damage  in  the 
top  fermented  beers  where  the  conditions  for  their  development 


BEER 


221 


(aeration)  is  more  favorable  than  in  the  bottom  fermented  beers. 
The  three  most  common  and  best  known  beer  acidifiers  are  Bac- 
terium aceti,  B.  pastorianus  and  B.  kutzingianum. 

Lactic  acid  and  butyric  acid  bacteria  may  gain  access  to  the 
fermenting  vats  and  render  the  beer  wholly  unfit  for  use.  Of 
these  two  kinds  of  bacteria,  the  butyric  acid  formers  are  by  far  the 
most  objectionable  because  of  the  very  disagreeable  odors  which 
they  form. 


Fig.  76. — Saccharomyces  elUpsoides.     The  common  wine  ferment.     Also  common  in 
jams,  jellies  and  canned  fruits. 


c.  Bitterness. — Bitterness  of  beer  may  be  caused  by  several 
species  of  so-called  wild  yeasts,  principally  Saccharomyces  pastori- 
anus I,  II  and  III,  and  of  these,  variety  I  is  the  most  common  and 
most  injurious.  It  is  stated  that  small  amounts  of  varieties 
II  and  III  are  not  objectionable  as  they  transmit  to  the  beer  a 
stronger  taste  and  smell. 

d.  Turbidity. — Turbidity  of  beers  may  be  the  result  of  a 
variety  of  factors  which  may  be  outlined  as  follows. 


222  BACTERIOLOGICAL  METHODS 

1.  Gluten  turbidity,  due  to  the  precipitation  of  protein 
substances. 

2.  Starch  turbidity,  due  to  the  presence  of  unchanged  starch. 

3.  Yeast  turbidity,  due  to  a  high  content  of  yeast  cells. 
If  wild  yeasts  are  the  cause  of  the  turbidity  then  there  may  be 
noticeable  a  bad  taste  and  bad  odor. 

4.  Bacterial  turbidity,  due  to  the  development  of  bacteria. 
In  this  case  there  may  be  noticeable  bad  odor,  bad  taste  and 
ropiness. 

5.  Sarcina  turbidity,  caused  by  the  members  of  the  sarcina 
group.  Unless  certain  species  are  present  in  large  numbers  the 
beer  may  not  be  appreciably  affected  in  quality.  It  must  be 
remembered  that  some  of  the  sarcinas  cause  disturbances  in 
gastric  digestion. 

3.  Wines. — Wine  is  grape  juice  which  has  undergone  alcoholic 
fermentation  through  the  action  of  yeast  organisms.  To  enter 
into  the  details  of  wine  production  is  not  necessary.  Wines  vary 
in  the  amount  of  alcohol  (8  to  16  per  cent.)  which  they  contain, 
in  color,  in  taste,  in  the  amount  of  unchanged  or  added  sugar, 
in  the  amount  of  acid,  etc.  Saccharomyces  ellipsoides  is  the  most 
common  yeast  concerned  in  the  alcohoHc  fermentation  of  grape 
juice.  Wine  diseases  are  frequently  met  with  and  are  not  unHke 
those  of  beer.  Ropiness,  turning  and  lactic  acidification  are 
perhaps  the  most  common,  induced  by  bacilli  and  cocci.  It 
may  be  stated  that  the  greater  natural  acidity  of  wines  in  general 
tends  to  retard  or  check  bacterial  invasion.  Bacterial  invasion  is 
also  in  a  measure  checked  by  the  greater  alcohol  content  of  wines 
over  that  of  beers.  Souring  of  wine  is  the  most  common  malady, 
induced  by  acetic  acid  bacteria  which  reduce  much  of  the  alcohol 
into  acetic  acid.  A  tough  membranous  scum  forms  on  the 
surface  of  the  wine,  composed  of  a  nearly  pure  culture  of  the 
acidifying  bacteria.  Bacterium  {Mycoderma)  aceti,  B.  pastorianum 
and  B.  kutzingianum  are  the  most  common  of  the  acid  formers 
and  the  three  species  appear  to  be  very  closely  related.     Souring  of 


WINE   AND    SAKE 


223 


wine  is  in  reality  a  normal  process  and  which  must  be  expected 
to  develop  under  ordinary  condition.  It  is,  however,  most  desirable 
to  retard  or  check  this  process  as  much  as  possible. 


Fig.  77. — Sak6.  Steamed  rice  cells  (c)  attacked  by  the  fungus  {Aspergillus 
oryzcs).  The  fungus  changes  the  starch  into  saccharine  substances.  Yeasts  and 
bacteria  are  usually  associated  with  the  hyphal  fungus,  feeding  upon  the  saccharine 
substances  formed. 


4.  Sake  or  Japanese  Rice  Wine. — Sake  is  a  fermented  drink 
quite  popular  in  Japan,  China  and  in  Corea.  It  is  made  from  rice 
which  has  been  steamed  to  soften  the  grain  and  starch  so  that 


224 


BACTERIOLOGICAL  METHODS 


the  fungus  aspergillus  oryzcB  may  convert  the  starch  into  saccharine 
compounds.     The  fungus  is  kept  on  hand  in  pure  culture  and 


Fig.  78. — Sak^.     Aspergillus  oryza,  showing  vegetative  hyphae  (a)  and  the  spore- 
forming  hyphae  {b,  c,  d). 

mixed  with  the  steamed  rice  and  the  sugar  fermentation  takes 
place  in  a  warm  room.  The  alcoholic  fermentation,  which  follows, 
is  much  like,  that  in  beer  making,  likewise  the  final  processes  of 


sake:  and  arrak 


225 


clarifying  and  pasteurizing.  This  drink  contains  from  14  to 
18  per  cent,  alcohol  and  is  essentially  a  wine.  It  may  be  taken 
cold  or  hot.  The  Japanese  usually  drink  it  hot.  There  are  several 
brands  of  sake  differing  in  quality.     There  is  a  sweet  variety 


Fig.  79. — Sake.  A,  Dead  or  dying  yeast  cells  (Saccharomyces  sake).  Vacuoles 
are  wanting,  the  cell  walls  are  generally  more  thickened  and  the  cells  are  somewhat 
shrunken  in  appearance;  B,  living  yeast  cells  showing  distinct  vacuoles;  C,  D, 
actively  budding  yeast  cells  (5.  sake)  and  hyphae  of  aspergillus  from  the  fermenting 
vats. 


(Mirin)  and  a  white  variety  (Shiro).     Sake  has  a  peculiar  aroma 
or  flavor  which  may  be  likened  to  that  of  bad  champagne. 

5.  Arrak. — This  Javanese  alcohoHc  drink  is  made  from  rice 
which  is  acted  upon  by  a  fungus  {Ragi)  similar  to  Aspergillus 


226  BACTERIOLOGICAL  METHODS 

oryz(B,  and  subsequently  the  alcoholic  fermentation  is  carried  on 
by  the  saccharomyces.  The  method  of  preparing  arrak  is 
therefore  similar  to  that  of  making  sake.  More  generally, 
however,  arrak  is  made  from  fermented  molasses. 

6.  Yoghurt. — This  is  Bulgarian  sour,  thick  or  klabbered 
sheep's  or  cow's  milk.  The  milk  is  boiled  and  evaporated  to  about 
half  its  volume,  then  cooled  to  about  45°  C.  and  the  ferment  known 
as  may  a  or  podkoassa  is  added.  The  may  a  is  simply  the  dry 
residue  from  a  previous  fermentation.  The  fermented'  product 
has  a  sour  aromatic  taste.     The  most  important  organism  in  this 


Fig.  80. — Showing  a  Kephir  granule  or  mass  natural  size  and  three  types  of  bacteria 
found  in  Kephir. — {Marshall.) 

fermentation  is  the  Bacillus  bulgaricus.  Other  bacilli,  cocci 
and  yeasts  are  also  present.  The  Yoghurt  tablets  of  the  market 
are  presumably  pure  cultures  of  the  Bacillus  bulgaricus. 

7.  Kephir. — Kephir  is  an  effervescent  alcoholic  sour  drink 
made  from  the  milk  of  the  cow,  sheep  or  goat.  This  is  also  a 
Bulgarian  preparation.  The  kephir  granules  or  seeds  are  simply 
more  or  less  dry  residues  of  a  previous  fermentation  and  may  be 
obtained  in  the  market.  These  granules  are  composed  of  the 
organisms  which  give  rise  to  the  fermentation  products,  principally 
Dispora  (Bacillus)  caucasica  and  several  species  of  streptococci. 
These  several  organisms  are  supposed  to  form  a  mutualistic  as- 


FERMENTED   MILKS 


227 


Sociation  and  cause  alcoholic  and  lactic  acid  fermentation  in  the 
imilk. 

8.  Koumiss. — This  drink  is  similar  to  kephir,  made  from 
mare's  milk,  by  the  inhabitants  of  southern  Russia  and  of  Siberia. 
The  active  organisms  in  the  ferment  are  a  yeast,  a  lactic  acid 
bacillus  and  a  second  species  of  bacterium  which  is  characteristic 
of  the  koumiss  and  which  appears  to  be  active  only  in  association 
with  the  other  organisms,  thus  also  indicating  a  mutualistic 
association.  The  fermented  milk  contains  lactic  acid  and 
alcohol. 

9.  Soja  Sauce. — This  Chinese  sauce  or  relish  is  made  from 
the  fermented  soja  bean  {Glycine  hispidus).     The  beans  are  boiled 


0      \<^^ 


<^ 


b^J 


_  ^ 


-:x/ 


^IG.  81. — Bacteria  of  slimy   wine.     A,  B,   C,  pure  cultures  of  various  forms;  D, 
mucilaginous  sheath  of  slime  bacteria.     {After  Kayser  and  Manceau.) 

md  mixed  with  parched  flour  and  then  exposed  to  the  ferment 
ispergillus  oryzce.  Salt  and  water  are  added  and  the  mixture 
s  allowed  to  ferment  slowly,  sometimes  for  years.  The  final 
Droduct  assumes  a  rich  brown  color  and  a  characteristic  aroma, 
.t  is  then  put  in  bags  and  almost  a  clear  juice  is  expressed  which 
s  then  further  clarified  and  pasteurized.  In  the  second  or  long 
)rocess  of  fermentation  several  organisms  are  active  along  with 
:he  Aspergillus,  as  Saccharomyces  soja,  Bacillus  soja  and  Sarcina 
lamayuchia. 

10.  Mazun. — This,  like  the  kephir  and  koumiss,  is  a  fermented 
nilk,  usually  of  the  cow  and  of  the  goat,  which  is  much  used  in 


228 


BACTERIOLOGICAL  METHODS 


Armenia.  The  active  organisms  in  the  ferment  are  a  bacillu! 
which  appears  to  be  identical  with  Bacillus  subtilis  and  also  severa 
different  kinds  of  lactic  acid  bacteria. 

II.  Leban. — This  sour  aromatic  drink  is  very  closely  similar  t( 
mazun  and  is  made  from  boiled  buffalo's,  cow's  and  goat's  milk 
It  is  of  Egyptian  origin.  It  is  said  to  contain  less  alcohol  than  doe: 
kephir.  Leban  fermentation  is  due  to  a  streptobacillus  whicl 
coagulates  milk  and  forms  lactic  acid.  A  diplococcus  is  als( 
present  which  ferments  glucose,  saccharose  and  maltose.     A  strep 


Fig.  82. — Sarcina  ventricttli. — (McFarland,  after  Migula.) 


tococcus  hydrolyzes  lactose  and  another  organism  is  capable  oj 
fermenting  glucose  and  maltose  but  not  lactose. 

12.  Ginger  Beer. — This  is  a  fermented  sugar  solution  to  whicl: 
ginger  has  been  added.  The  essential  fermenting  organisms  are 
a  saccharomyces  (5.  pyriformis)  and  Bacillus  vermijorme.  Myco 
derma  aceti  is  also  present.  The  two  essential  organisms  are 
evidently  in  close  mutualistic  relationship.  The  drink  produced 
is  acid  and  effervescing.  The  so-called  ginger  beer  plant  is  simpl> 
a  mass  or  matrix  of  the  active  organisms  and  is  used  for  the  purpose 


GINGER  BEER   AND  BEBEES 


229 


of  starting  the  fermentation.     The  ferment  is  evidently  closely 
related  to  the  following. 

13.  Bebee  Wine. — Bebees  or  California  Bees,  also  known  as 
Japanese  Beer  Seeds,  is  a  ferment  composed  largely  of  dried 
yeast  cells  which  when  added  to  solutions  of  sugar  or  molasses 
causes  a  quick  alcoholic  fermentation,  resulting  in  a  pleasant  alco- 
holic drink  (bebee  wine).  The  bebees  or  bebee  granules  resemble 
dried  peas  somewhat,  though  they  may  be  quite  variable  in 
size.  Some  15  years  ago  this  ferment  was  quite  common  in  the 
United  States,  having  been  reported  from  California,  Minnesota, 


^0  0  0  o 
.  0    '^-° 


J^o 


\  ^  0  ^  „    o  0          „ 


<=>iC3 


Pig.  83. — Vinegar  organisms.  A,  Bacterium  (Mycoderma)  aceti;  B,  Bacterium 
pasteurianus;  C,  Bacterium  kutzingianiim;  D,  B.  pasteurianum,  showing  the  mucilagi- 
nous sheath.  This  mucilaginous  material  causes  the  cells  to  stick  together  in  large 
masses,  forming  the  so-called  "mother  of  vinegar." — {Marshall.) 


Kentucky  and  other  states.  It  is  evidently  of  Japanese  origin. 
Kebler  and  Lloyd  made  brief  reports  on  this  ferment  several  years 
ago,  and  it  is  reported  that  the  ferment  has  disappeared  from  the 
American  market. 

Additional  products  of  fermentation  are  vinegar,  sauerkraut, 
pickled  cucumbers,  apple  cider,  yeast  cakes,  sour  dough  and  a 
lost  of  other  substances  used  as  food  or  employed  in  the  prepara- 
tion of  foods.  These  may  occasionally  come  to  the  notice  of  the 
"ood  bacteriologist.  Vinegars,  yeast  cakes,  sauerkraut  and  pickles, 
n    particular,  may    be    attacked    by    objectionable    organisms. 


230  BACTERIOLOGICAL   METHODS 

Diseases  may  enter  the  pickling  vats  and  ruin  the  entire  contents 
within  a  short  time.  "Hard  cider"  is  apple  wine  or  cider  in 
which  the  alcohol  has  been  largely  changed  into  acetic  acid  by  the 
My  coder  ma  aceti,  forming  cider  vinegar.  Cider  vinegar  in  turn 
may  be  invaded  by  bacteria  which  decompose  the  acetic  acid 
{Bacillus  xylenum). 

22.  Standardization  of  Disinfectants 

The  success  in  modern  surgery,  preventive  medicine  and 
sanitation  is  based  upon  the  use  of  disinfectants.  This  state- 
ment indicates  the  importance  of  disinfectants  as  articles  of  com- 
merce, suggests  the  necessity  of  adequate  supervision  of  the  manu- 
facture and  commercial  handling  of  these  substances  and  points 
out  the  necessity  of  guarding  against  adulteration  and  misrep- 
resentation. A  vast  array  of  so-called  antiseptics  have  been 
placed  on  the  market,  the  manufacturer  claiming  therefor 
properties  which  they  do  not  possess.  These  fraudulent  and 
exaggerated  claims  have  impelled  investigations  of  the  marketed 
disinfectants  with  a  view  to  determining  their  true  merit. 

Methods    for    standardizing    disinfectants    on    the    basis    of 
their  power  to  kill  or  destroy  bacteria  have  been  proposed  by 
various  investigators,  some  of  which  have  proven  quite  satis- 
factory.    The  Rideal- Walker  method  and  the  Lancet  method  of 
England  and  the  Anderson-McClintic  method  of  the  U.  S.  Public 
Health  Service  appear  to  find  most  favor,   the  latter  method  j 
being  a  modification  of  the  two  former.     In  the  Rideal-Walker  j 
and  Anderson-McClintic  methods  the  test  organism  used  is  the 
typhoid  bacillus,  exposing  definite  quantities  of  pure  cultures  of  j 
this  organism  to  varying  quantities  of  the  disinfectant  to  be 
tested  in  order  to  ascertain  the  kilHng  strength  as  compared 
with  the  standard  which  is  pure  phenol.     The  method  is  rather 
complicated  and  demands  great  care  and  precision  in  technique 
in  order  that  the  results  may  be  rehable  and  uniform  in  the  dif-  j 


STANDARDIZATION   OF  DISINFECTANTS  23 1 

ferent  laboratories.  A  simplified  method  will  no  doubt  be  sub- 
stituted for  the  Anderson-McClintic  method.  A  method  has 
been  proposed  based  on  the  percentage  of  bacteria  killed  within  a 
unit  of  time  by  a  unit  quantity  of  the  disinfectant  when  added 
to  a  unit  quantity  of  a  typhoid  bacillus  culture  known  to  contain 
a  definite  number  of  organisms.  The  Ohno-Hamilton  method 
is  simpler  than  the  Anderson-McClintic  method  and  is  included 
for  purposes  of  comparison. 

An  efficient  disinfectant  for  general  purposes  should  comply 
with  certain  requirements  which  may  be  stated  as  follows : 

1.  Should  be  highly  potent  as  destroyers  of  bacteria. 

2.  Should  be  readily  soluble  in  water  and  should  readily  permeate  or  penetrate 
solutions  of  organic  substances. 

3.  Should  be  comparatively  nontoxic  to  man,  when  applied  externally  or  when 
taken  internally. 

4.  Should  have  a  minimum  albumen  coagulating  power,  and  conversely  should 
be  capable  of  penetrating  organic  substances  readily. 

5.  Should  be  comparatively  cheap  and  should  be  readily  usable  by  those  of  aver- 
age ability  and  intelligence. 

The  ideal  disinfectant,  that  is,  one  which  is  highly  potent, 
readily  soluble  in  all  organic  solutions  and  capable  of  penetrating 
such  substances  readily,  and  at  the  same  time  nontoxic  and 
cheap,  does  not  exist.  There  is  no  disinfectant  which  is  highly 
efficient  as  a  destroyer  of  bacteria  and  at  the  same  time  non- 
toxic, notwithstanding  all  claims  to  the  contrary  by  manu- 
facturers. It  is  true,  however,  that  disinfectants  vary  greatly 
regarding  the  essentials  above  stated.  Our  present  means  for 
testing  the  efficiency  of  disinfectants  may  be  summarized  as 
follows : 

I.  John  F.  Anderson  and  Thomas  B.  McClintic^  of  the  United 
States  Public  Health  Service  have  worked  out  a  method  for  de- 
termining the  comparative  germ-destroying  power  of  disinfectants, 

^  John  F.  Anderson  and  Thomas  B.  McClintic.  A  Method  for  the  Bacteriolog- 
ical Standardization  of  Disinfectants.  The  Journal  of  Infectious  Diseases,  Vol. 
VIII,  No.  I,  Jan.  3,  191 1. 


234  BACTERIOLOGICAL  METHODS 


1 


Proportion  of  Culture  to  Disinfectant. — One-tenth  cc.  of  the 
culture  is  used,  added  to  5  cc.  of  the  disinfectant  dilution.  The 
amount  of  culture  is  measured  with  a  pipette  graduated  in  tenths 
of  a  cubic  centimeter.  fl 

Inoculation  Loops. — For  making  the  transfer  of  the  culture 
after  exposure  to  the  disinfectant  a  platinum  loop  4  mm.  in  diam- 
eter of  23  U.  S.  standard  gauge  wire  is  used.  We  have  found  it 
of  advantage  to  have  at  least  four,  and  preferably  six,  loops.  In 
order  to  save  time  in  flaming  the  following  method  was  devised : 

A  block  about  3  in.  wide,  10  in.  high,  and  12  in.  long,  containing 
four  or  six  grooves,  spaced  2  in.  apart,  is  used.  Into  each  of  the 
grooves  the  platinum  loop  is  laid  so  that  the  ends  of  the  loops 
extend  about  5  in.  beyond  the  side  of  the  block.  The  first  step 
in  the  operation  is  to  sterilize  each  loop  by  flaming  with  a  fantail 
Bunsen  burner  before  beginning  the  experiment. 

When  ready  to  begin  the  operation  the  loop  farthest  from  the 
operator  is  taken  in  the  right  hand  and  the  inoculation  made. 
It  is  then  replaced  in  the  groove  with  the  right  hand  and  the 
Bunsen  burner  (fan  tail)  placed  under  it  with  the  left  hand.  The 
next  loop  is  then  used,  replaced  in  its  groove,  and  the  Bunsen 
burner  placed  under  it  with  the  left  hand,  the  first  loop  having 
been  heated  to  redness  while  the  second  loop  was  in  use.  This 
procedure  is  then  continued  until  all  the  inoculations  have  been 
made.  The  time  required  in  making  the  inoculations  and  in 
replacing  the  loop  is  short,  it  being  found  that  15  sec.  is  ample. 

Incubations. — The  subcultures  are  incubated  45  hr.  at  37° 
C,  and  the  results  then  read  off  and  tabulated. 

Dilution. — Capacity  pipettes  for  the  original  dilutions  are 
invariably  used.  For  the  phenol  controls  a  standard  dilution 
of  pure  phenol  (Merck)  is  made  and  standardized  by  the  U.  S. 
P.  Method  (Koppeschaar)  to  contain  exactly  5  per  cent,  of  pure 
phenol  by  weight.  From  this  stock  solution  the  higher  dilutions 
are  made  fresh  each  day  for  that  day's  test. 

For  the  dilutions  of  the  disinfectant  a  5  per  cent,  solution  is 


STANDARDIZATION   OF  DISINFECTANTS  235 

made  by  adding  5  cc.  of  the  disinfectant  to  95  cc.  of  sterile 
distilled  water.  A  standardized  5  cc.  capacity  pipette  is  used 
for  this  and  after  filling  the  pipette  all  excess  of  the  disinfectant 
on  the  outside  of  the  pipette  is  wiped  off  with  sterile  gauze. 
The  contents  of  the  pipette  are  then  delivered  into  a  cylinder 
containing  95  cc.  of  sterile  distilled  water  and  the  pipette  washed 
out  as  clean  as  possible  by  aspiration  and  blowing  out  the  con- 
tents of  the  pipette  into  the  cylinder.  The  contents  of  the  cylinder 
are  then  thoroughly  shaken  and  the  dilutions  up  to  i  :  500  made 
from  it,  using  delivery  pipettes  for  measuring.  For  those  dis- 
infectants which  do  not  readily  form  a  5  per  cent,  solution  we 
make  a  i  per  cent,  stock  solution  and  from  this  make  the  dilutions 
greater  than  i  :  100  in  accordance  with  the  second  table  of  dilu- 
tions. If  greater  dilutions  than  i  :  500  are  to  be  made,  a  i  per  cent. 
solution  is  made  from  the  5  per  cent,  solution,  and  the  higher  dilu- 
tions made  from  this. 

We  had  adopted  the  following  scale  for  making  dilutions: 
For  dilutions  up  to  1:70,  increase  or  decrease  by  a  difference 
of  5  {i.e.,  5  parts  of  water). 

From  1:70  to  1:160  by  a  diflference  of  10 
From  1 :  160  to  i :  200  by  a  difference  of  20 
From  1:200  to  1:400  by  a  difference  of  25 
From  1 :  400  to  i :  900  by  a  difference  of  50 
From  1:900  to  i:  1800  by  a  difference  of  100 
From  i:  1800  to  1:3200  by  a  difference  of  200 

and  so  on  if  higher  dilutions  are  necessary. 

It  is  important  that  the  cylinders  used  for  making  the  dilutions 
be  correctly  graduated,  as  we  have  found  disregard  of  this  factor 
an  important  source  of  error.  It  is  preferable  to  use  standardized 
cylinders  and  pipettes,  and  we  recommend  that  they  be  used 
whenever  possible.  They  of  course  should  be  perfectly  clean. 
For  making  the  dilutions  in  accordance  with  the  above  scheme  we 
have  found  the  following  table  of  much  service: 


236 


BACTERIOLOGICAL  METHODS 


Table  I.    Stock  5  Per  Cent.  Solution,  (for  Dilutions) 

(5  cc.  disinfectant  +  95  cc.  distilled  water) 

Solution  A 


cc.  of  A 

CO.  Dist.  Water    j 

cc.  of  A 

cc.  Dist. 

Water 

cc.  of  A 

cc.  Dist.  Water 

i:  20 

20  +       0 

1 
or 

1 
10  + 

0 

1 
or 

44- 

0 

1:25 

20  +       5 

or 

10  + 

2-5 

or 

44- 

I 

1:30 

20  +     10 

or 

10  + 

5 

or 

A  4- 

2 

1:3s 

20  +      15 

or 

10  + 

7-5 

or 

4-t- 

3 

1:40 

20  +    20 

or 

10  + 

10 

or 

44- 

4 

1:4s 

20  +   25 

or 

10  + 

12.5 

or 

44- 

5 

1:50 

20  +  30 

or 

10  + 

15 

or 

4  + 

6 

1:55 

20+    35 

or 

10  + 

17-5 

or 

4-f 

7 

1:60 

20  +    40 

or 

10  + 

20 

or 

44- 

8 

i:6s 

20+    45 

or 

10  + 

22.5 

or 

4  + 

9 

1:70 

20  +    50 

or 

10+      • 

25 

or 

4  + 

10 

1:70 

20  +    50 

or 

10  4- 

25 

or 

4  + 

10 

1:80 

20  +    60 

or 

10  + 

30 

or 

4  + 

12 

1:90 

20  +    70 

or 

10  + 

35 

or 

4  + 

14 

i:  100 

20  +    80 

or 

10  + 

40 

or 

44- 

16 

1:110 

20  +    90 

or 

10  + 

45 

or 

44- 

18 

i:  120 

20  +  100 

or 

104- 

50 

or 

44- 

20 

1:130 

20  +  no 

or 

10  4- 

55 

or 

44- 

22 

1:140 

20  +  120 

or 

10  + 

60 

or 

4-1- 

24 

1:150 

20  +  130 

or 

10  + 

65 

or 

44- 

26 

1:160 

20  +  140 

or 

10  + 

70 

or 

44- 

28 

i:  160 

20  +  140 

or 

10  + 

70 

or 

44 

28 

1:180 

20  +  160 

or 

10  4- 

80 

or 

4  + 

32 

1:200  , 

20  +  180 

or 

10  + 

90 

or 

4  + 

36 

i:  200 

20  -f  180 

or 

44- 

36 

or 

24- 

18 

1:22s 

20  +  205 

or 

44- 

41 

or 

24- 

20.5 

1:250 

20  +  230 

or 

4-1- 

46 

or 

24- 

23 

1:275 

20  +  255 

or 

44- 

51 

or 

2-f 

25 -5 

1:300 

20  +  280 

or 

44- 

56 

*    or 

24- 

28 

1:325 

20  +  305 

or 

4  + 

61 

or 

24- 

30.5 

1:350 

20  +  330 

or 

4-f 

66 

or 

2-f 

32, 

1:375 

20  +  355 

or 

44- 

71 

or 

24- 

35-5 

1:400 

20  +  380 

or 

4-f 

76 

or 

2   + 

38 

1:450 

20  +  430 

or 

44- 

86 

or 

2   + 

43 

i:  "^oo 

20  4-  a8o 

or 

A   + 

06 

or 

2  4- 

^8 

STANDARDIZATION   OF   DISINFECTANTS 


237 


Table  II.    Stock  i  Per  Cent.  Solution  (for  Dilutions) 

(i  cc.  disinfectant;  99  cc.  distilled  water) 

Solution  A 


cc.  of  A 

cc.  Dist.  Water 

cc.  of  A 

cc.  Dist. 

Water 

cc.  of  A 

cc.  Dist.  Water 

I : 100  = 

TOO  +      0    or 

10  + 

0 

1:110  = 

100  +    10    or 

10  + 

I 

1:120  = 

100  +    20    or 

10  + 

2 

1 :  130  = 

100  +    30    or 

10  + 

3 

1:140  = 

100  +    40    or 

10  + 

4 

1:150  = 

100  +    50    or 

10  + 

5 

1:160  = 

100  +    60    or 

10  + 

6 

i:  160  = 

100  +    60    or 

10  + 

6 

[:i8o  = 

100  +    80    or  1 

10  + 

8 

[ :  200  = 

100  +  100    or 

10  + 

10 

:2oo  = 

100  +  100    or 

10  + 

10 

or 

4  + 

4 

:225  = 

100  +  125    or 

10  + 

12.5 

or 

4  + 

S 

:25o  = 

100  +  150    or 

10  + 

15 

or 

4  + 

6 

:275  = 

100  +  175    or 

10  + 

^7-5 

or 

4  + 

7 

'•300  = 

100  +  200    or 

10  + 

20 

or 

4  + 

8 

'325  = 

100  +  225    or 

10  + 

22.5 

or 

4  + 

9 

-350  = 

100  +  250    or  : 

10  + 

25 

or 

4  + 

10 

'375  =  ; 

100  +  275    or  , 

10  + 

27. S 

or 

4  + 

II 

:4oo  = 

100  +  300    or 

10  + 

30 

or 

4  + 

12 

:4oo  = 

10  +    30    or 

4  + 

12 

or 

2  + 

6 

:45o  = 

10  +    35    or 

4  + 

14 

or 

2  + 

7 

:50o  = 

10  +    40    or 

4  + 

16 

or 

2  + 

8 

:55o  = 

10  +    45    or 

4  + 

18 

or 

2  + 

9 

:6oo  = 

10  +    50    or 

4  + 

20 

or 

2+          ' 

10 

•  650  = 

10  +    55    or 

4  + 

22 

or 

2  + 

II 

:7oo  = 

10  +    60    or 

4+         1 

24 

or 

2  + 

12 

:75o  = 

10  +    65    or 

4+         ! 

26 

or 

2  + 

13 

:8oo  = 

10  +    70    or 

4+         ! 

28 

or 

2  + 

14 

:85o  = 

10  +    75    or 

4  + 

30 

or 

2  + 

15 

:90o  = 

10  +    80    or 

4  + 

32 

or 

2+          1 

16 

:9oo  =    j 

5  +    40    or  ^ 

4+         1 

32 

or 

2+          i 

16 

rooo  = 

5  +    45    or 

4+         j 

36 

or 

2+          1 

18 

1 100  = 

5  +    50    or 

4  + 

40 

or 

2  + 

20 

238 


BACTERIOLOGICAL   METHODS 


Table  II.    Stock  i  Per  Cent.  Solution  (for  Dilutions) 

(i  cc.  disinfectant;  99  cc.  distilled  water) 

Solution  A 


cc.  of  A 

cc.  Dist.  Water 

cc.  of  A 

cc.  Dist. 

Water 

cc.  of  A 

jcc.  Dist.  Watei 

1:1200  = 

5+    55    or 

4  + 

44 

or 

2   + 

22 

1:1300  = 

5  +    60    or 

4  +  - 

48 

or 

2  + 

24 

1:1400  = 

5  +    6s    or 

4-f- 

52 

or 

2  + 

26 

1:1500  = 

5  +    70    or 

4  + 

56 

or 

2  + 

28 

1:1600  - 

5  +    75    or 

4  + 

60 

or 

2  + 

30 

1:1700  = 

5  +    80    or 

4  + 

64 

or 

2  + 

32 

I : 1800  = 

5  +    85    or 

4  + 

68 

or 

2  + 

34 

1:1800  = 

5  +    85    or 

4  + 

68 

or 

2   + 

34 

1:2000  = 

5  +    95    or 

4  + 

76 

or 

2   + 

38 

1:2200  = 

5  4-  los    or 

4  + 

84 

or 

2   + 

42 

1:2400  = 

5  +  115    or 

4  + 

92 

or 

2   + 

46 

I : 2600  = 

5  +  125    or 

4  + 

100 

or 

2   + 

50 

1:2800  = 

5  +  135    or 

4  + 

108 

or 

2   + 

54 

1:3000  = 

5  +  145    or 

4  + 

116 

or 

2  + 

58 

1:3200  = 

5  +  155    or 

4  + 

124 

or 

2  + 

62 

Seeding  Tubes. — The  seeding  tubes  are  glass  test-tubes  i  in. 
in  diam.  and  about  3  in.  long,  with  round  bottoms.     In  order  to 


Fig.  84. — Block  for  holding  the  subculture  tubes. — (Anderson  b°  McClintic,  Hygiene 
Laboratory  Bulletin  No.  82,  U.  S.  Public  Health  Service  ) 

measure  the  disinfectant  into  them  they  are  placed  in  a  suitable 
wooden  stand  to  receive  them.  We  found  it  convenient  to  use  a 
wooden  block  containing  six  rows  of  fifteen  holes  each  for  the 
disinfectant  to  be  tested  and  a  separate  stand  for  the  phenol  con- 
trols. The  tubes  are  placed  in  the  stand  and  each  marked  with 
the  strength  of  dilution  it  is  to  contain.     The  rows  of  tubes  run- 


STANDARDIZATION  OF  DISINFECTANTS 


239 


ning  crosswise  represent  the  same  strength  dilution,  while  the  rows 
running  lengthwise  represent  the  different  strengths  to  be  used  in 
the  experiment. 

Starting  with  the  lowest 
dilution  (i.e.,  the  strongest), 
the  cylinder  is  shaken,  then  5 
tc.  are  measured  into  the 
tubes  of  the  row  to  receive 
that  strength,  using  a  5  cc. 
delivery  pipette.  In  order  to 
economize  glassware,  the 
same  pipette  is  used  for 
measuring  out  the  next  dilu- 
tion, first  blowing  out  as 
much  of  the  remaining  liquid 
as  possible,  then  drawing  a 
pipette  full  of  the  next  dilu- 
tion to  be  used  and  discard- 
ing that,  then  filling  the 
pipette  a  second  time,  which 
is  then  emptied  into  the  seed- 
ing tube. 

The  measuring  out  being 
completed,  the  tubes  are 
placed  in  the  water  bath  and 
allowed  to  stand  a  few  minutes 
in  order  that  the  disinfectant 
solution  may  reach  the  stand- 
ard temperature.  We  have 
not  found  it  necessary  to  use 
cotton  plugs  in  the  seeding 
tubes.  They  are  sterilized  in  paper-lined  wired  baskets,  with  the 
closed  end  of  the  tubes  up. 

Subculture  Tube  Racks. — Wooden  racks,  with  five  rows  of 


LHV^ILDER 


Fig.  85. — Water  bath  showing  position 
of  holes  for  the  thermometer  and  the  seeding 
tubes. — {Anderson  &  McClintic,  Hygiene 
Laboratory  Bulletin  No  82,  U.  S.  Public 
Health  Service.) 


240 


BACTERIOLOGICAL  METHODS 


fourteen  holes  each,  are  used  for  holding  the  subculture  tube 
and  as  plants  are  made  from  each  mixture  of  culture  and  di-s- 
infectant  every  2}^.  min.  up  to  15  min.,  six  tubes  are  required  for 
each  dilution.  Thus  in  each  rack  we  have  ten  rows  of  six  tubes 
each  with  two  empty  cross  rows  of  holes  left,  which  are  utilized 
by  placing  over  in  the  next  row  each  tube  as  it  is  planted.  This 
makes  it  easy  to  keep  run  of  the  tubes  that  are  planted.     It  is' 


r;,.;v;:.:;^.....v;,;,;..,....,,lii 


Fig.  86. — Cross  section  of  water  bath  showing  seeding  tubes  in  position. — 
{Anderson  &•  McCliniic,  Hygiene  Laboratory  Bulletin  No.  82,  U.  S.  Public  Health 
Service.) 


well  also  always  to  plant  from  the  seeding  tube  in  a  certain  hole 
in  the  water  bath  into  a  certain  row  of  tubes  in  the  rack.  This, 
after  a  Httle  practice,  will  help  to  avoid  errors  in  planting. 

Method  of  Conducting  the  Test. — If  there  are  in  one  experi- 
ment more  than  ten  dilutions  of  the  disinfectant,  including  the 
phenol  controls,  the  stronger  solutions  of   the  disinfectant  and 


STANDARDIZATION   OF  DISINFECTANTS  241 

phenol  are  tested  first,  as  it  will  not  be  necessary  to  plant  them 
after  7>^  min.  The  weaker  solutions  are  then  immediately  done 
and  are  planted  every  2}/^  min.  for  15  min. 

For  keeping  the  time  a  stop  watch  can  be  used,  but  an  ordinary 
watch  will  serve  the  same  purpose  by  simply  starting  on  the  2},^  or 
5  min.  periods. 

^  When  everything  is  in  readiness  the  culture  is  added  to  the 
disinfectant  solutions  with  a  sterile  pipette  in  tenths  of  a  cubic 
centimeter . 

To  add  the  culture,  the  seeding  tube  containing  the  disinfectant 
is  removed  from  the  water  bath  with  the  left  hand  and  slanted  at 
I  an  angle  of  about  45°,  and  with  the  right  hand  the  end  of  the 
pipette  containing  the  culture  is  introduced  and  lightly  touched 
against  the  side  of  the  tube  where  the  liquid  has  run  away  on 
account  of  slanting.  At  the  proper  time  the  culture  is  allowed  to 
run  into  the  disinfectant  solution,  the  pipette  removed,  the  tube 
straightened  up,  gently  shaken  three  times,  and  replaced  in  the 
water  bath.  The  other  tubes  are  done  the  same  way  in  succession, 
and  it  will  be  found  that  15  sec.  is  ample  time  for  each  tube.  By 
adding  the  culture  to  the  disinfectant  with  a  pipette  touched 
against  the  side  of  the  seeding  tube,  accurate  measurements  can 
be  made  and  each  tube  receive  exactly  the  same  amount  of 
''seeding,"  which  is  not  the  case  when  the  culture  is  added  by  the 
"drop." 

If  ten  tubes  are  to  be  inoculated,  only  a  few  seconds  will  remain 
after  inoculating  the  last  tube  before  a  plant  from  the  first  tube 
will  have  to  be  made. 

The  mixing  tubes  are  not  removed  or  disturbed  in  making  the 
planting  except  to  insert  the  loop  or  spoon  into  them,  touch  the 
bottom,  withdraw,  and  then  make  the  plant  in  broth.  Every 
effort  is  made  to  insert  and  withdraw  the  loops  and  spoons  in  a 
uniform  manner.  The  loops  and  spoons  are  bent  to  an  angle  of 
about  45°  where  they  are  joined  on  to  the  shank,  and  therefore  are 
always  filled  with  the  mixture  when  withdrawn  from  the  seeding 


242 


BACTERIOLOGICAL   METHODS 


tubes.  After  making  the  plants,  the  loops  or  spoons  are  flamed  ai 
already  described. 

After  an  experiment  is  finished  the  date  and  any  necessan 
details  can  be  marked  on  one  of  the  broth  tubes  and  the  racl 
placed  in  the  incubator  at  37°  C.  for  48  hr.  At  the  end  of  thi 
time  the  results  are  recorded  on  a  chart  especially  devised  for  th( 
purpose.     (See  Table  III.) 

Determining  the  Coefficient. — After  a  large  number  of  experi 
ments,  we  have  concluded  that  the  method  employed  by  theLance 


faU=.. 


Fig.  87. — Device  for  holding  and  flaming  inoculating  loops. — (Anderson  br  McClintic 
Hygiene  Laboratory  Bulletin  No.  82,  U.  S.  Public  Health  Service.) 

Commission,  with  certain  modifications,  is  the  best  one  for  de 
termining  the  coefficient,  i.e.,  the  mean  between  the  strength  anc 
time  coefficients. 

In  performing  the  test,  plants  are  made  every  2}4  i^in.  up  t( 
and  including  15  min.  To  determine  the  coefficient,  the  figur( 
representing  the  degree  of  dilution  of  the  weakest  strength  of  th( 
disinfectant  that  kills  within  2j^  min.  is  divided  by  the  figun 


STANDARDIZATION   OF  DISINFECTANTS 


243 


representing  the  degree  of  dilution  of  the  weakest  strength  of  the 
phenol  control  that  kills  within  the  same  time.  The  same  is  done 
for  the  weakest  strength  that  kills  in  1 5  min.  The  mean  of  the  two 
is  the  coefhcient.  The  method  of  determining  the  coefficient  will 
be  seen  in  Table  III. 


Date:  May  18,  1913. 


TABLE  III 

Name,  "A." 

Temperature  of  medication,  20°  C. 

Culture  used,  B.  typhosus;  24  hr.;  extract  broth  filtered. 

Proportion  of  culture  and  disinfectant,  o.i  cc.  +  5  cc. 

Organic  matter,  none;  kind,  none;  amount,  none. 

Subculture  media,  standard  extract  "broth.     Reaction,  +  1.5;  quantity  in  each  tube, 

10  cc. 


Time  Culture  Exposed  to  Action  of  Disinfectant 

for  Minutes 

Sample 

DUution 

Phenol  Coefficient 

2M 

3 

l\i 

10 

X2\^ 

IS 

Phenol 

1:80 

— 

_ 

— 

1:90 

+ 

— 

— 

— 

i:  100 

+ 

+ 

+ 

— 

-       — 

— 

1:110 

+ 

+ 

+ 

+ 

X 

— 

Disinfec- 

1:350 

— 

— 

— 

tant  "A" 

1:375 

- 

- 

- 

1:400 

+ 

~ 

— 

80)375(4-69 

1:425 

+ 

+ 

- 

- 

- 

- 

110)650(5.91 

1:450 

+ 

+ 

_ 

- 

- 

- 

2)10.60 

1:500 

+ 

+ 

— 

— 

— 

— 

S-30 

1:550 

+ 

+ 

+ 

— 

— 

— 

1:600 

+ 

+ 

+ 

+ 

— 

— 

1:650 

+ 

+ 

+ 

+ 

+ 

— 

1:700 

+ 

+ 

+ 

+ 

+ 

+ 

5.30  =  phenol 

1 :  750 

+ 

+ 

+ 

+ 

+ 

+ 

coeflEicient 

To  Determine  the  Comparative  Cost  per  Unit  of  Efficiency. — 

W'hen  bids  are  solicited  for  supplying  disinfectants  they  should 
3e  required  to  be  raade  so  as  to  show  the  comparative  cost  per 
[oo  units  of  efficiency  of  the  disinfectant  as  compared  with  100 
mits  of  pure  phenol.  It  is  manifestly  cheaper  to  purchase  a 
17 


244 


BACTERIOLOGICAL  METHODS 


disinfectant  that  sells  for  60  cents  a  gallon  than  one  that  sells  for 
30  cents  a  gallon,  if  the  former  has  four  times  the  efficiency  of  the 
latter. 

The  true  cost  of  a  disinfectant  can  be  determined  only  by 
taking  into  consideration  the  phenol  coefficient  and  the  cost  per 
gallon  of  the  disinfectant. 

The  following  table  (IV)  is  a  good  illustration  of  the  value 
of  a  determination  of  the  comparative  cost  per  100  units  of 
disinfectant  in  terms  of  100  units  of  pure  phenol: 

Table  IV 


Disinfectant 

Phenol  Coefficient 

Price  per  Gallon 

Relative  Cost  per  loo 
Units  of  Efficiency  as 
Compared  with  Pure 
Phenol 

Car 

2.12 

4.44 
1.40 

1. 13 
0.44 
0.2s 
I.OO 

$0.30 
I.OO 

0.37 
0.44 

0.41 
0.40 
2.67 

5.2 

Chi 

8.4 

Phi..                  

9.9 

Cre 

14-5 

Nap.      .           

34.8 

Zod 

59-6 

Pure  phenol 

100. 0 

It  will  be  seen  that  the  substance  Chi  has  a  higher  coefficient 
than  any  of  the  others  in  the  table,  but  its  high  cost  per  gallon 
results  in  its  being  placed  second  in  cost  per  100  units. 

The  cost  per  100  units  of  efficiency  as  compared  with  pure 
phenol  is  obtained  by  first  dividing  the  cost  per  gallon  of  the] 
disinfectant  by  the  cost  per  gallon  of  pure  phenol;  this  give 
the  price  ratio  between  the  disinfectant  and  pure  phenol;  th( 
cost  ratio  is  then  divided  by  the  phenol  coefficient,  which  gives  u! 
the  cost  per  unit  of  efficiency  as  compared  with  pure  phenol  =  i 
The  cost  per  unit  is  then  multiplied  by  100  to  give  the  cost  pel 
100  units. 

The  Ohno-Hamilton  Phenol  Coefficient 

Tatsuzo  Ohno  and  H.  C.  Hamilton  of  the  Parke,  Davis  Re 
search  Laboratory  have  proposed  a  method  for  the  bacteriologies 


STANDARDIZATION  OF  DISINFECTANTS  245 

standardization  of  disinfectants,  which  is  a  decided  simplifica- 
|tion  of  the  Anderson-McClintic  (U.  S.  Public  Health  Service) 
i  method,  and  it  is '^hereby  given  in  somewhat  abbreviated  form 
!  (American  Journal  of  Pubhc  Health,  May,  191 2). 
I  I.  The  organism  used  is  a  vigorous  culture  of  B.  typhosus 
grown  for  24  hr.  in  standard  bouillon  culture  medium  at  38°  C. 
It  is  taken  from  the  incubator  at  least  K  hr.  before  using,  to 
allow  gradual  adjustment  to  changed  conditions  of  temperature 
before  exposure  to  the  germicide.  The  culture  and  germicidal 
agent  should  always  be  at  the  same  temperature  before  interac- 
tion takes  place. 

The  24  hr.  bouillon  culture  is  removed  from  the  incubator 
and  kept  at  room  temperature  without  agitation  for  about  half 
an  hour.  Then,  without  shaking  the  culture,  as  is  usually  done, 
it  is  decanted  into  a  specially  constructed  cotton  filter,  thus  leav- 
ing scum  and  large  clumps  on  the  filter  and  filtering  the  individual 
Dacteria  in  a  practically  isolated  state.  It  is  then  filtered  into 
1  sterile  test-tube,  which  is  subsequently  shaken  in  order  to  ob- 
tain a  homogeneous  filtrate  and  make  it  ready  for  use. 

The  cotton  filter  for  the  filtration  of  bacterial  cells  is  an  or- 
iinary  test-tube  drawn  out  at  one  end  Kke  a  centrifugal  tube,  the 
miali  end  cut  open  and  the  edges  smoothed  with  a  flame.  Into 
;he  large  end  a  small  pledget  of  good  quality  ordinary  cotton  is 
ntroduced  as  far  as  the  constricted  portion  of  the  tube,  pushed 
gently  with  the  forceps,  taking  care  not  to  form  any  fissure  in 
;he  cotton  or  to  leave  any  spaces  between  the  cotton  and  tube. 
The  open  end  of  the  tube  is  then  plugged  with  cotton  and  the 
vhole  wrapped  in  cotton  and  parchment  paper  and  sterihzed  by 
Iry  heat  as  usual.  Before  using  the  cotton  filter  after  steriliza- 
ion,  the  cotton  in  the  tube  should  be  gently  pushed  back  to  the 
)roper  place  by  means  of  a  sterile  pipette,  so  that  it  is  in  exactly 
he  same  position  as  before  sterilization.  During  sterilization  the 
otton  is  pushed  up  by  the  tension  caused  by  the  heat  and  its  own 


246  BACTERIOLOGICAL  METHODS 

elasticity,  producing  an  undesirable  space  between  the  cotton  and 
the  constricted  part  of  the  tube. 

II.  Culture  Medium. 

500  grams  chopped  beef. 
20  grams  peptone  (Witte). 
5  grams  sodium  chloride. 
1000  cc.  water. 

The  beef  is  digested  at  50°  C.  for  M  br.,  then  boiled,  strained, 
the  other  ingredients  added,  then  boiled  again,  filtered  and  ad- 
justed to  +'  I  reaction.  \ 

III.  The  dilutions  of  sample  and  standard  are  made  eitherjbyj 
weight  or  volume,  depending  on  the  character  of  the  disinfectant] 
to  be  tested.  In  the  case  of  liquids  such  as  the  coal-tar  disin- 
fectants, both  sample  and  standard  should  be  diluted  by  volume. 

The  Sample. — Dilutions  of  an  emulsive  coal-tar  product 
should  be  made  by  adding  water  gradually  to  the  measured 
quantity  of  disinfectant.  The  reason  for  this  is  that  in  some 
cases  the  character  of  the  emulsion  is  greatly  altered  by  the 
method  of  making  the  dilution. 

An  emulsion  is  less  likely  to  break  if  it  is  made  as  follows! 
To  make  a  i  per  cent,  solution,  moisten  the  measuring  flask  01 
cylinder  with  about  2  cc.  water.  With  a  capacity  i  cc.  pipette 
measure  the  disinfectant  and  mix  it  with  the  water,  using  this 
mixture  for  a  partial  cleaning  of  the  pipette.  Then  add  more 
water,  stirring  just  enough  to  mix  but  not  to  make  the  mixtun 
foam.  Wash  out  the  pipette  by  drawing  up  and  expelling  th< 
dilution,  then  make  up  to  the  mark. 

This  method  requires  more  care  in  measuring  the  final  diluj 
tion  if  the  meniscus  is  obscured  by  the  emulsion.     If  the  lasj 
addition  of  water  is  made  by  carefully  running  it  down  the  si< 
of  the  container,  the  surface  liquid  will  not  greatly  obscure 
reading. 

This  I  per  cent,  solution  is  further  diluted  to  the  desired 


STANDARDIZATION   OF   DISINFECTANTS  247 

tent  by  mixing  with  distilled  water  in  proper  proportion,  in  each 
case  adding  the  measured  disinfectant  to  the  measured  quantity 
of  water  to  make  the  desired  dilution. 

The  Standard. — Merck's  pure  phenol  is  diluted  by  volume 
by  weighing  out  any  desired  quantity,  dividing  by  the  specific 
gravity,  1.08,  and  dissolving  in  distilled  water,  diluting  to  twenty 
times  the  volume  of  the  carbolic  acid  used,  to  make  a  5  per  cent, 
solution  by  volume  in  volume.  Further  dilutions  can  be  made 
from  this  as  desired,  since  the  solution  is  practically  permanent. 

The  dilutions  of  carbolic  acid  ordinarily  used  with  the  results 
to  be  expected  are  as  follows: 


■Minutes 

Dilutions 

I 

2 

3 

4 

5 

I-IIO 

+ 

.  — 

— 

— 

— 

1-120 

4- 

+ 

+ 

— 

— 

1-130 

+ 

+ 

+ 

'+ 

- 

The  sample  is  usually  diluted  in  such  a  way  that  not  more 
than  a  half  unit  in  the  coefficient  lies  between  two  dilutions. 
For  example 

1-480         1-540         1-600         1-660         1-720 

the  intention  being  to  compare  with  the  carbolic  acid  dilution 
1-120.  A  disinfectant  value  based  on  growth  at  i  min.  only 
would  be  far  from  exact.  Much  more  accurate  results  can  be 
obtained  where  the  dilutions  compared  are  those  which  kill  the 
organism  in  from  3  to  5  min. 

IV.  The  proper  mixture  of  culture  and  disinfectant  is  carried 
out  as  follows :  five  drops  of  bacterial  filtrate  are  introduced  into 
5  cc.  of  germicidal  solution,  contained  in  test-tubes  53-^  in.  long 
by  %  in.  in  diam.  This  is  added  drop  by  drop  in  rapid  suc- 
cession, by  means  of  a  sterile  pipette,  83^  in.  long  and  0.219  in. 
in  diam.,  the  narrow  end  measuring  about  0.075  in.  in  diam., 
held  vertically  at  the  mouth  of  the  test-tube  containing  the 
germicidal  solution,  so  that  the  pipette  with  the  germs  will  not 


248  BACTERIOLOGICAL   METHODS 

come  into  direct   contact  with  any  part  of   the   test-tube  con- 
taining the  germicidal  solution.     If  any  of  the  organisms  adhere 
to  the  sides  or  to  threads  of  cotton  in  the  mouth  of  the  tube  they 
might  be  inoculated  into  the  bouillon  without  being  exposed  to 
the  germicide.     As  soon  as  the  last  one  of  the  five  drops  is  added 
to  the  germicidal  solution,  they  are  mixed  thoroughly  for  15  sec. 
or  longer  by  holding  the  test-tube  in  the  left  hand  and  shaking  it ; 
with  the  fingers  of  the  right  hand;  a  formation  of  air  bubbles  ; 
shaped  like  a'^long  funnel  extending  from  the' bottom  of  the  tul 
toward  the  surface  of  the  liquid  shows  that  the  mixing  is  efli 
ciently  done,  thus  bringing  every  bacterial  cell  into  direct  contiu  1 
with  the  germicidal  solution. 

V.  The  subcultures  are  taken  each  minute  for  5  min.  by  means 
of  a  23  platinum  wire  loop  of  4  mm.  inside  diam. 

Tests  of  two  dilutions  can  be  carried  on  at  the  same  time  b\ 
one  person,  as  a  half-minute  is  more  than  is  ordinarily  necessai 
for  taking  out  a  subculture  and  planting  into  the  tube  of  cultui 
medium.     Tests  of  five  dilutions  of  a  sample  and  three  of  tli* 
standard  can  therefore  be  made  in  about  25  min. 

To  obtain  a  loopful  of  the  mixture,  the  test-tube  should  !> 
tilted  so  as  to  prevent  getting  the  foam  formed  on  the  surface  01 
the  mixture  during  shaking  instead  of  getting  a  liquid  portion  (  1 
the  mixture.     The  wire  loop  should  be  plunged  almost  to  tl; 
bottom  of  the  tube  before  withdrawing. 

These  loopfuls  are  inoculated  into  test-tubes  containing  about 
6  cc.  of  the  standard  culture  medium   above  described.     The  \ 
are  placed  in  the  incubator  at  38°  C.  for  48  hr.  or  for  such 
time  as  it  is  found  that  no  further  development  of  bacteria  tak( 
place. 

VI.  Conclusions  as  to  the  value  of  a  disinfectant  from  a  tesi 
conducted  as  described  are  drawn  by  comparing  the  dilutions  of 
the  sample  and  the  standard,  which  are  equally  efficient.  While 
in  general  one  dilution  of  each  is  used  for  comparison,  it  is  often 
necessary  to  take  the  mean  of  a  number,  since  it  is  not  uncommon 


STANDARDIZATION   OF  DISINFECTANTS  249 

for  two  dilutions  to  give  practically  identical  results.     Note,  for 
example,  such  results  as  these: 


Carbolic  acid 

1-120 

+ 

+ 

—         — 

1-130 

+ 

+ 

+ 

Sample 

1-720 

+ 

+ 

—         — 

1-780 

+ 

+ 

-         - 

i-840 

+ 

+ 

+         + 

In  this  case  if  no  other  dilutions  of  the  sample  are  tested  the 
value  is  determined  by  comparing  both  720  and  780  with  carbolic 
acid  dilute  120  and  the  result  is  6.25,  the  average  of  the  two 
values. 

It  will  be  noted  that  in  the  foregoing  description  no  mention 
is  made  of  the  temperature  at  which  the  test  is  made.  While 
temperature  affects  very  vitally  the  process  of  disinfection,  the 
changes  in  temperature  of  an  ordinary  working  room  rarely 
exceed  10°  C,  while  the  average  change  would  not  exceed  5°  C, 
the  year  round;  and  since  the  standard  is  affected  to  practically 
the  same  degree  by  these  changes  it  seems  an  unnecessary  com- 
plication to  carry  out  the  test  at  a  rigidly  defined  temperature. 

It  will  also  be  noted  that  the  time  of  contact  between  organism 
and  disinfectant  is  only  one-third  as  long  as  is  recommended  in 
most  tests.  While  it  may  form  in  some  cases  a  better  picture  of 
the  value  of  a  disinfectant  to  find  its  efficiencies  at  two  periods  such 
as  2j^  min.  and  15  min.,  practically  no  material  change  in  its 
value  results  from  such  a  course.  The  taking  of  a  subculture 
each  minute  rather  than  at  2j^  min.  intervals  makes  for  greater 
accuracy,  but  this  does  not  materially  affect  the  results. 

The  Worth  Hale  Toxicity  Coefficient 

Worth  Hale  of  the  U.  S.  Public  Health  Service  Hygienic 
Laboratory  has  worked  out  a  method  for  determining  the  com- 
parative toxicity  of  disinfectants  of  which  the  following  is  a 
briefly  summarized  outline. 


250  BACTERIOLOGICAL  METHODS 

Test  Animals. — The  animals  upon  which  the  substance  in 
question  is  to  be  tested  shall  be  white  mice  of  not  less  than  15 
nor  more  than  30  grams  weight. 

Dilutions  and  Dosage. — The  dose  is  to  be  calculated  per  gram 
of  body  weight  and  should  when  diluted  equal  between  0.03  and 
0.04  cc.  per  gram  weight;  that  is,  0.6  to  0.8  cc.  for  a  20  gram  mouse. 
The  diluent  is  to  be  distilled  water  and  primary  dilutions  are  to 
be  made  of  such  strength  that  the  dose  is  easily  measured  with 
a  I  cc.  pipette  graduated  into  hundredths.  This  is  most  easily 
accomplished  by  the  use  of  the  substance  in  greater  concentra- 
tion than  is  required  to  kill  in  the  above  volume  doses. 

Administration  of  the  Test  Solutions. — After  the  required 
dose  of  the  diluted  disinfectant  has  been  estimated  it  is  meas- 
ured into  a  suitable  dish  and  is  then  diluted  further  to  the  re- 
quired volume  by  adding  sterile  distilled  water  in  sufficient  quan- 
tity. A  series  of  mice  are  then  injected  subcutaneously  with 
varying  amounts  of  the  substance  until  the  least  fatal  dose  (L. 
F.  D.)  is  determined,  the  mice  being  kept  under  observation. 

Time  Limit  of  the  Observation. — After  the  animals  have  been 
inoculated  they  are  kept  under  observation  for  a  period  of  24  hr. 
unless  death  results  in  a  shorter  period  of  time. 

Phenol  Comparative  Test. — Mice  of  the  same  lot  are  similarly 
injected  with  pure  phenol  properly  diluted  to  make  the  meas- 
urements of  the  dose  easy  and  then  further  diluted  in  a  small 
dish  to  equal  a  volume  dose  of  0.03  to  0.04  cc.  per  gram  of  body 
weight  and  the  fatal  dose  determined  as  above.  This  least  fatal 
dose  (L.  F.  D.)  of  phenol  is  unity  and  the  least  fatal  dose  of 
the  substance  in  question  is  estimated  in  per  cent,  of  this. 

Determining  the  Comparative  Toxicity. — The  phenol  toxicity 
of  the  disinfectant  tested  is  to  the  toxicity  of  phenol  as  x  is  to  100. 
The  example  given  below  would  be  represented  in  the  following 
proportions:  4.5  :  18  ::x :  100  =  25  per  cent.,  that  is  disinfectant 
''A"  is  one-fourth  as  toxic  as  is  pure  phenol. 


STANDARDIZATION  OF   DISINFECTANTS 
Table  V 


251 


Name  of  Disinfectant 

Mouse, 
Weight 

Dose  per  Gm., 
Body  Weight 

Result 

Time.  Hr., 
Min. 

Disinfectant  "A" 

21.13 
20.64 
18.32 
19.05 

18.46 
20. 10 
19.23 
18.90 

0.0012 
0.0016 
0.0018 
0.0020 

0.0035 
0 . 0040 
0.0045 
0 . 0050 

Survived 

Survived 

Died 

Died 

Survived 

Survived 

Died 

Died 

Pure  Phenol 

10:30 
2:15 

1:15 
0:25 

Valuable  information  regarding  the  comparative  toxicity  of 
many  substances  used  as  disinfectants  may  be  obtained  from 
a  study  of  the  comparative  medicinal  doses.  For  example,  the 
medicinal  doses  of  phenol,  betol,  resorcinol  and  corrosive  sub- 
limate are  i  grain,  3  grains,  4  grains  and  Mo  grain  respectively. 
These  doses  are  practically  in  proportion  to  the  toxicity  of  the 
substances  nam.ed  and  stating  the  dosage  in  the  terms  of  the 
phenol  toxicity  coefficient  as  proposed  by  Hale,  we  would  get  the 
following  results: 


Phenol .  .  .  . 

Betol 

Resorcinol. 


100.00 

33-30 

25  00 

Corrosive  sub 3000 .  00 


As  a  rule,  however,  the  exact  composition  of  many  of  the 
proprietary  disinfectants  is  either  not  made  known  to  the  users 
or  is  not  disclosed  by  the  manufacturers  and  in  such  cases  the 
only  thing  to  be  done  in  order  to  ascertain  whether  or  not  the 
claims  of  the  manufacturers  are  correct,  is  to  make  tests  as  above 
outUned.  However,  in  cases  where  the  composition  of  the  "dis- 
infectant is  definitely  known,  whether  a  simple  or  compound 
substance,  its  comparative  toxicity  can  be  determined  by  as- 
certaining the  toxicity  of  the  several  ingredients  and  rating  in 
comparison  with  the  standard,  namely,  pure  phenol. 


252  BACTERIOLOGICAL   METHODS 

The  Albumen  Coagulating  Coefficient  of 
Disinfectants 

As  is  well  known  to  surgeons  and  pathologists,  the  action  of 
disinfectants  and  their  value  in  tissue  disinfection  and  in  the 
disinfection  of  organic  matter  such  as  sputum,  excreta,  etc., 
varies  according  to  their  albumen  coagulating  power.  Some 
disinfectants,  as  alcohol,  mercuric  chloride,  silver  nitrate,  copper 
sulphate  and  others,  coagulate  albumen  very  actively  and  this 
property  checks  or  prevents  further  penetration  and  action. 
Furthermore,  inert  combinations  between  the  coagulating  dis- 
infectants (metallic  ions)  and '  the  albuminous  substances  are 
formed  which  render  a  portion  of  the  disinfectant  unavoidable  for 
further  action.  This  behavior  explains  why  some  disinfectants  are 
more  active  when  diluted,  as  for  example  alcohol  and  carboHc  acid. 
Even  high  dilutions  of  copper  sulphate  (i  :  50,000  to  i  :  4,000,000) 
will  gradually  kill  bacteria  in  water  or  in  other  nonorganic  liquids, 
due  to  a  coagulation  of  the  bacterial  plasm,  whereas  solutions  of 
5  per  cent,  to  10  per  cent,  of  the  same  substance  are  considered 
rather  unsatisfactory  disinfectants.  The  stronger  solutions 
coagulate  the  albuminous  matter  in  which  the  bacteria  may  be 
imbedded,  no  doubt  quickly  kiUing  the  organisms  in  the  exposed 
outer  layers  of  the  albuminous  particles  or  masses  while  the 
layer  of  coagulum  encloses  many  of  the  bacteria  effectually  pro- 
tecting them  against  further  action  of  the  disinfectant.  These 
enclosed  bacteria  may  become  liberated  after  a  time  due  to  a 
breaking  up  of  the  coagulated  covering  or  coating  and,  if  patho- 
genic, may  cause  a  single  infection  or  an  epidemic. 

The  following  are  some  of  the  disinfecting  agents  which  pre- 
cipitate or  coagulate  albumen  actively: 

Alcohol.  Chloral  hydrate. 

Ether.  Phenol. 

Salts  of  heavy  metals.  Picric  acid. 

Camphor.  Mineral  acids. 

Volatile  oils.  Some  organic  acids. 
Tannic  acid. 


STANDARDIZATION   OF   DISINFECTANTS  253 

The  following  are  disinfecting  agents  which  do  not  precipitate 
or  coagulate  albumen: 

Acetic  acid.  Salts  of  light  metals. 

Phosphoric  acid.  Lysol. 

Alkalies  and  soaps.  Cresols. 

While  the  albumen  coagulating  power  of  the  different  disin- 
fectants varies  greatly,  it  does  not  follow  that  a  disinfectant  which 
coagulates  albumen  actively  in  strong  solution  will  do  so  when  in 
weaker  solution.  For  example,  pure  carbolic  acid  is  a  strong 
coagulant  but  in  solutions  of  5  per  cent,  and  less  it  is  indeed  a  very 
weak  coagulant.  It  is  therefore  not  exactly  in  accord  with  fact  to 
designate  carbolic  acid  as  a  disinfectant  having  a  high  coagulating 
power  and  hence  comparatively  unsuitable  as  a  tissue  (abscesses, 
infected  wounds,  etc.),  and  organic  matter  (ejecta,  excreta,  etc.), 
disinfectant,  because  in  strengths  of  2.5  per  cent,  and  5  per  cent, 
it  has  only  a  slight  coagulating  power,  but  is  still  very  active  as  a 
germ  (nonsporebearing)  destroyer. 

It  is  not  intended  to  imply  that  a  disinfectant  becomes  useless 
as  soon  as  it  begins  to  coagulate  albumen  actively,  but  the  indica- 
tions are  that  the  noncoagulating  disinfectants  are  more  satis- 
factory than  those  which  are  active  coagulants.  The  coagulating 
coefficients  give  that  solution  strength  of  the  disinfectants  tested, 
which  indicates  or  marks  a  retardation  in  disinfecting  efficiency 
due  to  the  coagulation  of  albuminous  matter.  This  albumen 
coagulating  coefficient  is  wholly  independent  of  the  germ  destroy- 
ing coeiSicient  as  well  as  that  of  the  toxicity  coefficient. 

The  following  is  an  outline  of  the  proposed  method  for  de- 
termining the  comparative  albumen  coagulating  power  of  disin- 
fectants, at  the  same  time  also  indicating  the  solution  percentage 
limit  of  optimum  efficiency  and  usefulness  as  disinfectants. 

Albumen  Test  Solution 

The  standard  test  solution  shall  be  a  i  per  cent,  aqueous 
(distilled  water)  solution  of  pure  dried  egg  albumen. 


2  54  BACTERIOLOGICAL  METHODS 

The  following  methods  for  making  the  albumen  solution  are 
submitted. 

I.  Gravimetric  Method  A. — Place  2  grams  of  pure  powdered 
egg  albumen  in  100  cc.  of  boiled  distilled  water,  shake  and  set 
aside  for  6  to  12  hr.,  shaking  frequently.  Filter  through  a 
tared  filter  paper  which  has  been  dried  (at  100°  C.)  to  constant 
weight.  Filtering  is  slow,  requiring  perhaps  i  hr.  time. 
When  the  last  drop  has  filtered  through,  dry  the  filter  paper, 
with  the  unfiltered  albumen  residue  upon  it,  to  constant  weight 
and  weigh.  Deduct  from  this  weight  the  weight  of  the  dried  filter 
paper  to  obtain  the  weight  of  the  albumen  residue.  From  i 
gram  of  egg  albumen  dried  to  constant  weight  determine  the 
percentage  of  moisture.  From  the  data  thus  obtained  it  is  easy 
to  determine  the  amount  of  boiled  distilled  water  which  must  be 
added  to  the  filtrate  (100  cc.)  to  make  i  per  cent,  dried  albumen 
solution. 

We  will  suppose  that  the  dried  filter  paper  to  be  used  in  filtering 
the  albumen  solution  weighs  1.570  grams  and  this  same  paper  with 
the  undissolved  albumen  residue  (also  dried  at  100°  C.  to  constant 
weight)  weighs  1.965  grams,  then  the  weight  of  the  undissolved 
dried  albumen  residue  equals  0.395  gram.  We  will  suppose  that 
I  gram  of  albumen  loses  0.126  gram  on  drying,  or  12.6  per  cent, 
moisture.  0.395  gram  raised  to  its  normal  air  moisture  (0.395 
gram  +  12.6  per  cent  of  0.395  gram  =  0.444  gram)  and  subtracted 
from  2.00  grams  leaves  1.556  grams,  the  amount  of  albumen  that 
passed  through  the  filter  paper.  12.6  per  cent,  of  1.556  grams  = 
0.196  gram,  and  1.556  grams  less  0.196  gram  =  1.360  grams  which 
represents  the  amount  of  albumen,  dried  to  constant  weight,  that 
passed  into  solution.  Therefore  to  make  a  i  per  cent,  solution  it 
is  necessary  to  add  enough  boiled  distilled  water  to  the  filtrate  to 
make  i:  100.  In  this  case  add  water  up  to  the  136.00  cc.  mark. 
We  now  have  a  i  per  cent,  solution  sufficiently  accurate  for  all  prac- 
tical purposes. 

This  albumen  test  solution  is  now  ready  for  use  but  it  must  b( 


STANDARDIZATION   OF   DISINFECTANTS  255 

kept  in  mind  that  it  is  readily  attacked  by  microbes.  However, 
if  carefully  prepared  with  pure  albumen,  boiled  distilled  water,  in 
sterile  vessels,  and  put  on  ice  or  in  a  cool  place,  it  will  keep  for 
perhaps  4  days. 

Any  quantity  of  albumen  solution  may  be  made,  it  merely 
being  advised  not  to  prepare  more  than  may  be  required  for  the 
tests  contemplated. 

2.  Gravimetric  Method  B. — In  a  dried  and  tared  platinum  dish 
place  5  cc.  of  the  albumen  filtrate  (2  grams  in  100  cc.  of  boiled 
distilled  water),  evaporate  over  water  bath  and  dry  to  constant 
weight,  and  from  this  determine  the  percentage  of  albumen  in  the 
solution  and  the  amount  of  water  that  must  be  added  to  the 
albumen  filtrate  to  make  i  per  cent. 

The  Phenol  Standards 

The  standard  of  comparison  is  the  opacity  produced  in  5 
cc.  of  the  I  per  cent,  egg  albumen  solution  when  5  cc.  of  5  per 
cent,  phenol  solution  are  added  (in  a  standard  test-tube  of  about  15 
cc.  capacity).  This  phenol  tube  is  placed  against  a  black  back- 
ground. In  making  a  test,  varying  dilutions  of  the  disinfectant 
are  added  to  the  egg  albumen  solutions  in  a  series  of  test-tubes 
until  the  opacity  produced  is  the  same  as  that  in  the  phenol  tube. 
In  each  test  5  cc.  of  the  dilution  are  added  to  5  cc.  of  egg  albumen 
in  a  standard  test-tube  and  the  two  tubes  compared,  placed  against 
a  black  background. 

Dilutions  of  Disinfectants  to  be  Tested 

The  phenol  control  solution  (5  per  cent.)  is  made  as  for  the 
Anderson-McClintic  method  of  standardizing  disinfectants, 
using  only  pure  phenol  crystals. 

Of  the  disinfectants  to  be  tested,  10  per  cent,  and  i  per  cent, 
primary  stock  solutions  are  made;  10  per  cent,  solutions  of  liquid 


256  BACTERIOLOGICAL  METHODS 

disinfectants  as  alcohol,  formalin  and  acids,  and  i  per  cent, 
solutions  of  the  salts  of  heavy  metals  and  of  soluble  substances 
generally.  From  these  primary  •  stock  solutions  the  following 
secondary  dilutions  or  substock  solutions  are  made,  always  in 
those  amounts  which  will  serve  the  purpose,  that  is,  in  amounts 
for  perhaps  ten  subdilutions  for  each  and  every  disinfectant  to 
be  tested. 

I  :  10  (of  Hquids  only.) 

1 :  100 

I : 1000 

1 :  10,000 

1 :  100,000 

Method  of  Testing 

1.  Phenol  Standard. — Pour  5  cc.  of  the  egg  albumen  solution 
in  a  standard  test-tube,  using  a  standard  5  cc.  pipette  having  a 
free  outflow.  Add  to  this  5  cc.  of  the  phenol  stock  solution  (5 
per  cent.).  Set  tube  in  the  standard  test  rack  (with  black  back- 
ground made  of  cardboard  covered  with  black  tissue  paper). 
The  degree  of  opacity  developed  is  to  serve  as  the  standard  of 
comparison. 

2.  Preliminary  Testing. — The  albumen  coagulating  power  of 
the  disinfectant  being  unknown,  much  time  and  labor  can  be 
saved  by  testing  with  the  four  or  five  substock  solutions,  adding  5  cc. 
to  5  cc.  of  the  egg  albumen  test  solution,  in  order  to  find  that  dilu- 
tion of  the  disinfectant  which  fails  to  show  any  opacity.  We  will 
suppose  that  the  i  :  1000  substock  solution  shows  very  marked 
opacity  or  precipitation,  then  the  i  :  10,000  solution  might  be  tried, 
which  may  also  show  quite  marked  opacity,  then  the  i  :  100,000 
may  be  tried.  If  this  gives  negative  results  then  we  know  that 
the  phenol  standard  lies  between  i  :  10,000  and  i  :  100,000  with  the 
probabilities  that  it  is  nearer  i  :  10,000. 

3.  Concluding  Testing.^ — Going  back  to  the  i  :  10,000  dilution, 
make  ten  subdilutions,  increasing  the  dilutions  by  a  difference  of 


STANDARDIZATION   OF  DISINFECTANTS  257 

1000    by  simply  adding  the  required  parts  of    distilled  water, 
using  small  quantities,  thus: 

10  parts  of  I  :  10,000  +  i  part   water  =  i  :  11,000 
10  parts  of  I  :  10,000  +  2  parts  water  =  i  :  12,000 
10  parts  of  I  :  10,000  +  3  .parts  water  =  i  :  13,000 
Etc. 

Any  other  quantity  proportions  may  be  used,  however,  as 
10,  15,  20,  etc.,  parts  of  the  substock  solution  with  the  required 
parts  of  distilled  water.  If  the  i  :  looo  substock  solution  is  to 
be  used  then  the  dilutions  should  be  increased  by  loo,  as  follows: 

10  +  I  =  I  :iioo 
10  +  2  =  I  :  1200 
10  +  3  =  I  :i30o 
10  +  4  =  I  :  1400 

or  any  other  equal  proportion  of  stock  solution  and  distilled  water 
may  be  used,  as 

5  +  0.5,  100  +  10,  or  1000  +  100,  etc. 

If  the  highest  stock  dilution  (i :  100,000)  is  to  be  used,  then  the 
increase  should  be  10,000,  thus: 

10  +  I  =  I  ;  110,000 
10  +  2  =  I  :  120,000 
10  +  3  =  I  •  130,000 

Determining  the  Phenol  Coefficient 

Having  determined  that  dilution  which  gives  the  same  coagula- 
tion opacity  as  the  5  per  cent,  carbolic  acid,  it  is  a  very  simple 
matter  to  determine  the  phenol  albumen  coagulating  coefficient 
by  simply  dividing  the  strength  of  the  dilution  of  the  disinfectant 
tested  by  the  phenol  dilution  (1:20). 

The  following  are  the  coagulating  coefficients  of  a  few  dis- 
infectants: 


258 


BACTERIOLOGICAL   METHODS 


Name  of  Disinfectant 


Reaction  Limit 


Phenol  Coefi&cient 


Phenol 

Copper  sulphate 

Mercuric  chloride 

Silver  nitrate 

Alcohol  (95  per  cent.) 


I  :  20 

I  :  15,000 
I  :  10,000 
I  :  9,500 
I  '3 


1. 00 

750.00 

500 . 00 

475- 00 

0.15 


The  following  table  gives  the  efficiency  value  of  some  dis- 
infectants. It  will  be  seen  that  this  value  is  of  necessity  variable, 
depending  upon  the  variation  in  the  market  price  of  the  disinfec- 
tants. It  will  also  be  seen  that  in  the  proposed  rating  the  re- 
markably high  coagulation  coefficient  of  some  of  the  more  im- 
portant chemical  disinfectants  lowers  the  efficiency  value 
greatly. 

Efficiency  Values  of  a  Few  Disinfectants 


Name  of  Dis- 
infectant 


Phenol 
Coeflf. 


Tox. 
Coefif. 


Coat 
Coefl 


Comp. 
Cost 


Eff. 
Value 


Special 
Properties 


Phenol 

Boracic  acid. 


Chlorornaphtho- 

leum 
Copper  sulphate . . 

Lysol 

Mercuric  chloride. 

Neko 


1. 00 
0.23 
6.06 
330 


43.00 


1. 00 
0.05 
0.16 
1 .00? 
0.4S 
50 


Potassium        per- 
manganate 
Silver  nitrate 


20 .  00    i     o .  20 


0.85    I     0.50 


Trikresol. 


38.00 

2.62 


300 

0.90 


750 


650 


475 


015 
1. 00 

015 
1. 00 

015 
1. 00 
0.20 
1 .20 
0.65 

4-33 
1.27 
8.46 
0.50 

3-33 
0.25 
1.66 
564 
37 -60 
0.40 
2.66 


1. 00 

o.  20 

5.22 

0.004 

0.44 

0.06 

5-66 

0.04 

0.075 

0-73 


Odor 

Odorless 

Odor 

Odorless.     Slight 

color 
Odorless 

Odorless,    Corrodes' 

metal 
Odor 

Odorless.      Deodor- 
ant.   Stains 
Odorless.     Stains 

Odor 


STANDARDIZATION   OF  DISINFECTANTS 


259 


The  efficiency  value  of  any  disinfectant  is  found  by  dividing 
the  phenol  coefficient  by  the  sum  of  the  other  coefficients,  as 
follows : 

Phenol  coefficient  Efficiency 

Tox.  coefficient  +  coag.  coefficient^  comp.  cost  "^     value 

In  the  table  the  first  figure  in  the  comparative  cost  column  is 
the  market  price  per  pound  of  the  disinfectant  and  the  second  figure 
is  the  comparative  cost  (compared  with  phenol  at  15  cents 
per  pound) . 

The  Toxicity  and  Germ  Destroying  Power  of  Some  of 
THE  More  Important  Disinfectants 

The  values  given  are  obtained  from  various  sources  and  in  some 
instances  require  further  verification.  The  table  will  serve  as  a 
?uide  to  a  valuation  of  the  disinfectants  for  purposes  of  general 
disinfection. 

Germ  Destroying  Power  and  Toxicity  of  Disinfectants 


Name 


Ucohol 

Uum 

Immonia 

Lmmonium  chloride. 
JLmmonium  sulphate . 


Liitozone , 

Lrsenious  acid. . 
iirsenite  of  soda. 

tacterol 

ienetol 


ichloride  of  platinum. . 

loracic  acid 

romine 

Cabot's  sulpho-naphthol. 
alcium  chloride 


18 


Germ  Destroying  Power 

Toxicity 

(Phenol  as  i) 

(Phenol  as  loo) 

0.03 

S-OO 

0.64 

10.00 

2.40 

15.00 

0.03 

10.50 

0.015 

500 

0.00 

0.50 

5000 . 00 

0-33 

3000.00 

1.58 

45.00 

1.23 

33  00 

10.00 

0.23 

5-00 

S-oo 

3.87 

11.00 

0.08 

3-50 

26o  BACTERIOLOGICAL  METHODS 

Germ  Destroying  Power  and  Toxicity  of  Disinfectants — Continued 


Name 


Germ  Destroying  Power  ; 
(Phenol  as  i) 


Camphor 

Carbolene 

Carbolic  acid 

Carbolozone 

Car-sul 

Caustic  acid 

Chinosol 

Chloride  of  gold 

Chlorine 

Chloro-naphtholeum 

Chromic  acid 

Copper  sulphate 

Corrosive  sublimate 

Cre-bol-you 

Cremolene 

Creo-carboline  disinfectant 

Creola 

Creoleum  (Dusenberry). . . 

Creolin  (Pearson) 

Creolol  (Rudish's) 

Creosol  (saponified) 

Creo-Sul 

Creosoleum 

Cresylone 

Crude  carbolic  acid 

Cupric  chloride 

Cyllin 

Dioxygen 

Electrozone 

Ether 

Ferrous  sulphate 

Formacone  liquid 

Formaldehyde 


1.36 
1. 00 
1.48 
2.00 


4- 

20 

II 

.00 

0 

.02 

0 

.90 

2 

•50 

0 

27 

0 

.04 

0 

30 

Toxicity 
(Phenol  as  100) 


33-30 

11.00 

100.00 

6,40 

16.00 


0.17 

120.00 

0.95 

25.00 

12.50 



12.50 

6.06 

16.00 

15.00 

100 . 00 

3-30 

100.00? 

43.00 

3000 . 00 

9.00 

1.26 

403 

30.00 

0.52 

12.80 

1 .00 

9.00 

3  25 

18.00 

1.24 

13.00 

1.03 

6.40 

15.00 

2.90 

11.00 

56.00 

2-75 

90.00 

80.00 


25.00 

0.50 
40.00 

75.00 


STANDARDIZATION  OF  DISINFECTANTS  26 1 

Germ  Destroying  Power  and  Toxicity  op  Disinfectants — Continued 


Nam« 


Germol 

Glycerin  (sp.  gr.  1.25) 

Hycol 

Hydrate  of  chloral .  .  .  . 
Hydrocyanic  acid 


Hydrogen  peroxide. 

Hygeno  A 

Iodine 

Iron  sulphate 

Izal 


Killitol.. 
Kreosota. 
ECreotas. . 

Kreso 

Kresolig . 


fCretol 

ead  chloride 

.ead  nitrate 

.incoln  disinfectant. 

iquid  creoleum 


.iq.  cres.  comp.,  U.S.P. 

.isapol 

isterine 

3^sol 

/Tercuric  chloride 


lercuric  iodide 

lilkol 

iineral  acids 

[aphthalene 

Faphthol  phenoline. 


Feko 

Titrate  of  cobalt 

oncarbolic  disinfectant. 


Germ  Destroying  Power 
(Phenol  as  i) 


•   2 

.12 

0 

.015 

12 

•30 

0 

•32 

7 

•SO 

6 

•30 

3 

.56 

12 

•50 

0 

.27 

8 

.00 

0 

02 

I 

26 

I 

10 

3 

92 

2 

18 

0 

92 

I 

SO 

0 

83 

I. 

48 

3- 

00 

0. 

01 

2.12 
43  00 


[-1.50 
2.50 
6.40 

20.00 
1.50 


Toxicity 
(Phenol  as  100) 


16 

.00 

0 

50 

32 

00 

10 

00 

10,000 

00 

5 

00 

17 

00 

400 

00 

40 

00 

5.60 

5 -60 
22.50 
56.00 

14.00 
200.00 
300.00 

17.00 
9.00 

56.00 

5000 

0.20 

45.00 

5000 . 00 

1000.00 

11.00 

I 20 . 00 

750 
6.40 


7.50 


262 


BACTERIOLOGICAL   METHODS 


Germ  Destroying  Power  and  Toxicity  of  Disintectants — Continued 


Name 


Germ  Destroying  Power 
(Phenol  as  i) 


Toxicity 
(Phenol  as  100) 


Osmic  acid 1 

Phenaco i 

Phenol  (pure) 

Phenol  disinfectant 

Phenol,  disinfecting  and  cleansing  . .  | 

Phenol  liquid,  U.S.P.  (1890) 

Phenol  sodique 

Phenosote 

Phenotas  disinfectant 

Pi-ne-ex 

Pino-lyptol 

Piatt's  chlorides 

Potassium  bichromate 

Potassium  cyanide 

Potassium  iodide 

Potassium  permanganate 

Public  health  disinfectant 

Pyxol 

R.  R.  Roger's  disinfectant 

Salicylate  of  soda 

Salicylic  acid 

Sanax 

Sanitas 

Saponified  cresol 

Silver  nitrate 

Sodium  borate 

Sodium  chloride ; 

Sulpho-naphthol j 

Tarola i 

Trikresol i 

20th  Cent,  disinfectant 

Veriform  germicide 

Victor  sanitary  fluid 


20.00 

15.00 

1 .00 

0.61 


1.77 
o.oi 
3-43 
1-37 


1000 . 00 

32.00 

100.00 

7-50 

80.00 
4.50 

19.00 
9.00 

10.00 


0.27 

3.20 

O.OI 

3.00 

500 . 00 

3.00 

1500.00 

0.02 

5.00 

0.85 

25.00 

0.48 

28.00 

3 -03 

56.00 

3.20 

6.50 

0.30 

5.00 

0.22 

22.00 

0.30 

6.00 

1.03 

300.00 

38  00 

5.00 

0.04 

00.15 

0.02 

00.05 

3.S7 

3.12 

16.00 

2.62 

90.00 

0.13  . 

10.00 

0-43 

15.00 

13.00 

STANDARDIZATION   OF   DISINFECTANTS  263 

Germ  Destroying  Power  and  Toxicity  oe  Disinfectants. — Continued 


f..   ^  I  Germ  Destroying  Power 

Name  i  (Phenol  as  i) 


Wescol  disinfectant , 

Worrel's  disinfectant o.oi 

Zenoleum 

Zinc  chloride 

Zodane 


2.25 
1.56 
0.04 


Zodone  (4) 1.62 

Zonol 2.37 


Toxicity 
(Phenol  as  100) 


19 

00 

100 

00 

8 

60 

10 

00 

The  Narcotic  and  Antiseptic  Properties  of  the  Essential 

Oils 

It  is  generally  believed  that  the  addition  of  spices  to  foods 
serves  to  preserve  them,  that  is,  prevent  decomposition  changes. 
In  a  general  way  this  is  in  accord  with  facts.  The  antiseptic 
properties  of  essential  oils  are  quite  marked  and  the  antiseptic 
properties  of  spices  are  largely  due  to  the  essential  oils  which  they 
contain.  Important  investigations  in  regard  to  the  narcotic  and 
antiseptic  properties  of  the  more  important  essential  oils  have 
been  made  by  Martindale,  Coupin  and  Geinitz.  The  following 
is  a  summary  of  results  by  Geinitz  as  given  in  the  Semi-annual 
Report  of  Schimmel  and  Co.,  for  Oct.,  191 2. 

The  principal  outcome  of  Geinitz'  investigations  is  the  establishment  of  the  fact 
j  that  the  narcotic  and  disinfecting  properties  of  the  essential  oils  do  not  correspond 
with  those  of  the  active  constituents  of  those  oils;  the  sequence  of  the  series  differs 
widely.  For  example,  Russian  anise  oil  and  its  active  constituent,  anethol,  have 
j  no  antiseptic  action  whatsoever,  but  both  have  a  pronounced  narcotic  action  upon 
cold-blooded  animals.  It  would  appear  that  the  group  which  exerts  an  antiseptic 
action  and  that  which  acts  narcotically  are  not  found  in  the  same  molecule  of  the 
odoriferous  bodies;  nay,  in  many  of  these  substances  one  of  the  groups  is  wanting 
altogether.  It  is  also  necessary  to  abandon  the  theory  that  narcosis  is  determined 
simply  by  the  great  solubility  of  lipoid  in  the  cells  of  the  nervous  system,  and  that 
the  antiseptic  action  of  essential  oils  depends  upon  solubility  of  the  bacteria  in  the 
lipoids.  The  explanation  of  the  facts  which  have  been  observed  is  probably  that 
the  organism  of  the  bacteria  with  its  peculiar  metabolic  process  occupies  in  Nature  a 


264  BACTERIOLOGICAL  METHODS 

position  wholly  for  itself.  For  the  results  of  narcotic  experiments  which  have  been 
obtained  with  essential  oils  in  the  case  of  coid-blooded  animals  and  in  that  of  the  higher 
plants  are  altogether  dififerent  from  those  which  have  been  obtained  with  bacteria. 

The  results  of  narcotic  experiments  with  fishes  and  tadpoles,  of  respiration 
experiments  with  toads  and  of  injection  experiments  with  frogs  are  reproduced  in 
the  form  of  tables  arranged  according  to  the  degree  of  activity  of  the  essential  oils. 

For  the  purpose  of  testing  narcotic  action  on  fishes,  roaches  {Leuciscus  rutilus)  were 
used.  The  limit  of  concentration  taken  was  the  dilution  which  produced  perceptible 
narcosis  in  the  fish  within  a  period  of  24  hr.,  that  is  to  say,  a  condition  when  the 
animal,  without  displaying  much  spontaneous  motion,  floated  in  the  water  in  an 
atactic  condition  and  altogether  failed  to  respond  to  squeezing  with  hooked  pincers. 
Only  those  experiments  were  regarded  as  affording  proof  in  which  the  fish  recovered 
when  replaced  in  fresh  water. 

For  the  purpose  of  estimating  the  antiseptic  action,  Geinitz  added  in  each  case 
to  10  cc.  of  fresh  milk,  placed  in  a  test-tube  of  16  to  18  cc.  capacity,  first  as  much 
Sulphur  depuralum  as  would  lie  on  the  point  of  a  knife,  and  afterward  the  antiseptic. 
After  vigoro  us  shaking  a  piece  of  filtering  paper  soaked  with  solution  of  lead  acetate 
was  hung  up  in  the  upper  part  of  the  test-tube  in  such  a  way  as  not  to  come  in  contact 
with  the  milk,  and  the  test-tube  was  closed  with  a  wad  of  cotton-wool.  The  tubes 
were  then  kept  24  hr.  in  a  water  plug  bath  at  about  38°  C.  If,  after  that  lapse  of 
time,  the  lead  paper  was  found  to  be  blackened,  it  was  evident  that  the  tube  in 
question  did  not  contain  a  sufficient  proportion  of  the  antiseptic.  As  a  series  of  test- 
tubes  was  always  being  treated  with  an  increasing  quantity  of  antiseptic,  it  was  easy 
to  determine  exactly  when  the  limit  of  concentration  was  reached  at  which  the  activ- 
ity of  the  bacteria  was  impeded.' 

Experiments  in  Narcosis,  Made  on  Fishes 


Substance                           Dilution 

Substance                            Dilution 

Mustard  oil  {aWyliso- 

sulphocyanate) 

Cinnamon  oil 

I  :  1,320,000 
180,000 
153,846 
134,010 

177,7,77, 

Fennel  oil. . . .                           i  •  ^j.  ic^i; 

Terpineol  (liquid) 32,000 

Coumarin 28  571 

Citral 

Carvacrol 

Thyme  oil 

Turpentine  oil,  fraction 

containing  /3-pinene 28,000 

Carvone 

Sandalwood  oil 

125,918    Borneo  camphor 26,087 

116,327    EucalvntoWCine.olV                       00  nor. 

Eugenol 

Anethol 

111,836 
104,587 

/-a-Pinene 20,105 

^  Abstracted  from  the  Sitzungsherichte  und  abhandlungen  der  naturjorschenden 
Gesellschaft  zu  Rostock,  New  Series,  Vol.  IV,  191 2.  Rostock,  1913.  The  paper  was 
awarded  a  prize. 

The  evolution  of  sulphuretted  hydrogen  from  milk  diluted  with  sulphur  is  due  to 
bacterial  action. 


STANDARDIZATION   OF   DISINFECTANTS 
Experiments  in  Narcosis,  Made  on  Fishes — Continued 


265 


Substance 


Dilution 


Greek  turpentine  oil 86,405 

French  turpentine  oil 79A93 

Spanish  turpentine  oil 77>304 

American  turpentine  oil 77>304 

Calamus  oil 71,862 

German  peppermint  oil 70,000 

Russian  anise  oil 67,000 

Clove  oil 67,000 

Mitcham  peppermint  oil 66,666 

Caraway  oil 62,500 

Safrol 60,560 

Heliotropin 45A54- 

Heptylalcohol 45,000 

Rose  oil 41,715 

Lavender  oil 38,764 

Mace  oil 38,527 

Octylalcohol 38,090 

Geraniol 37, 500 


Substance 


Benzaldehyde 

Oenanthol 

Menthenone 

Umbellulone 

Eulimene     (artificial 

limonene) 

Rosemary  oil 

Juniperberry  oil 

Cymene 

Terpineol  (cryst.) . . . 

Anisic  aldehyde 

Lemon  oil 

Chloroform , 

Fenchyl  wo  valerate. . , 
Bornylwovalerate. .  . . 
Limonene 

Chloral  hydrate 

Alcohol. 

Ether 


Dilution 


18,096 
16,788 
16,000 
15,000 


13,000 

13,333 
11,320 

10,965 
io,SS4 
10,164 

9,157 
8,070 

6,345 
6,300 
1,040 

286 
190 
166 


23.  Determining  the  Purity  and  Quality  of  Sera,  Bacterins  and 

Related  Products 


Sooner  or  later  the  regulatory  work  under  the  pure  drugs  laws 
of  the  land  will  cover  the  newer  remedies  which  have  come  into 
prominence  within  recent  years,  such  as  therapeutically  active 
sera,  the  so-called  bacterial  vaccines  or  bacterins,  tuberculins, 
smallpox  vaccine,  rabies  vaccine,  glandular  extracts,  etc.  It 
is,  however,  self-evident  that  such  supervision  on  the  part  of  the 
bacteriologists  in  drugs  laboratories  will  not  be  necessary  as  far 
as  the  products  manufactured  under  supervision  of  the  U.  S. 
Public  Health  Service  are  concerned.  State  and  city  authorities 
(inspectors)  may  perhaps  find  a  supply  of  these  products  in  the 


266  BACTERIOLOGICAL   METHODS 

more  remote  drug  stores  which  have  exceeded  the  age  limit  or 
which  have  become  deteriorated  in  some  manner,  but  even  this 
must  be,  in  the  very  nature  of  things,  rather  a  remote  possibility. 
It  is  therefore  not  likely  that  the  drug  bacteriologist  will  be 
called  upon  to  examine  any  of  the  standard  products  put  up  in 
the  Government  inspected  laboratories.  There  are,  however, 
numerous  preparations  placed  on  the  market  which  are  said  to 
have  properties  similar  to  the  standard  sera,  etc.,  but  which  are 
of  a  fraudulent  character.  It  then  becomes  necessary  to  resort 
to  certain  tests  which  will  determine  whether  or  not  the  article 
under  consideration  possesses  the  properties  claimed  for  it.  Such 
tests  are  both  chemical  and  bacteriological.  The  chemical  tests 
are  largely  qualitative  and  include  certain  color  reactions,  pre- 
cipitation reactions,  etc.  However,  much  remains  yet  to  be  done 
in  the  way  of  devising  methods  which  will  prove  practically  useful, 
Some  of  the  very  recent  laboratory  guides  to  the  examination  of 
medicinal  substances  contain  suggestions  which  will  prove  useful, 
and  these  may  be  applied  in  special  cases.  For  example,  a  number 
of  chemical  tests  have  been  suggested  for  determining  the  presence 
of  ductless  gland  products  and  of  various  animal  secretions.  The 
absolute  merit  of  these  tests  is  seriously  questioned  by  some  authori- 
ties; however,  their  confirmatory  significance  is  generally  admitted. 

Biological  products  the  activity  of  which  depends  upon  the 
presence  of  living  germs  are  comparatively  few  and  are  not  likely 
to  be  brought  to  the  attention  of  the  drug  bacteriologist.  The  bio- 
logical products  are  intended  for  hypodermic,  intravenous,  intra- 
muscular or  some  similar  mode  of  use  and  must  therefore  conform 
to  certain  specific  requirements.  They  must  be  entirely  free  from 
all  undesirable  foreign  bacteria,  dead  or  alive,  and  must  not  con- 
tain undesirable  foreign  biological  or  toxicological  products. 

The  complete  examination  of  biological  products  comprises 
standardization  and  certain  so-called  safety  tests,  and  is  carried 
out  in  all  of  the  laboratories  operating  under  Government  super- 


BIOLOGICAL   PRODUCTS  267 

vision.     These  tests,  as  carried  out  in  the  laboratories  of  Parke, 
Davis  and  Co.,  may  be  outlined  as  follows: 

I.  Standardization. 

1.  Potency. — Determining  the  number  of  units  per  cc. 

2.  Activity. — Ascertaining  the  power  to  produce  the  desired  results.  This 
is  simply  a  check  on  the  potency  test. 

3.  Serum  Tests. — In  some  of  the  biological  products  certain  tests  are  made 
to  determine  the  difference  between  anti-sera  and  the  normal  serum  of  the 
same  species. 

II.  Safety  Tests. 

1 .  Freedom  from  bacterial  contamination  in  those  products  which  are  sup- 
posedly free  from  living  germs. 

2.  Determining  the  purity  of  the  cultures  in  those  products  which  are  com- 
posed of  pure  cultures  of  a  given  kind  of  germ. 

3.  In  case  of  products  which  are  supposed  to  contain  dead  bacteria  only, 
tests  are  made  to  determine  the  absence  of  all  organisms  capable  of 
multiplying. 

In  order  to  test  biological  products  as  to  the  absence  of  viable 
or  living  organisms,  about  2  cc.  of  the  sample  is  cultured  under 
aerobic  and  anaerobic  conditions.  To  determine  the  purity  of  a 
! product  containing  Hving  bacteria,  cultures  are  made  in  suitable 
media  and  these  are  carefully  studied  as  to  specific  cultural  char- 
acteristics and  appearance  under  the  compound  microscope. 

For  the  purpose  of  standardizing  the  products,  a  well-equipped 
llaboratory  is  necessary,  including  the  necessary  experimental 
animals.  The  full  routine  followed  out  in  the  laboratory  of  the 
factory  need  not  be  carried  out  in  the  regulatory  drug  laboratory. 
In  most  cases  the  work  will  consist  of  making  animal  inoculation 
tests  to  determine  the  potency  of  the  marketed  article  in  order  to 
ascertain  whether  or  not  it  possesses  the  properties  claimed  for  it. 
In  some  instances  it  may  be  necessary  to  determine  the  presence 
of  toxic  ingredients.  Perhaps  the  most  likely  tests  will  be  those 
which  come  under  the  head  of  potency  and  safety  tests.  For  the 
present  purpose  the  above  outline  will  no  doubt  suffice.  As  to 
what  methods  may  become  desirable  and  necessary,  only  time 
and  further  experience  will  indicate. 

! 


268  BACTERIOLOGICAL  METHODS 

The  bacterial  contamination  of  smallpox  vaccine  has  received 
considerable  attention  on  the  part  of  American  bacteriologists.  \ 
Such  vaccines  are  rarely  wholly  free  from  extraneous  bacteria,  no : 
matter  how  carefully  prepared.     It  is  rather  remarkable  that  the 
method  of  manufacturing  the  vaccine  is  not  modified  in  accordance 
with  modern  progress  in  sanitation.     Since  the  smallpox  virus  is 
filterable  it  would  seem  possible  to  pass  the  dissolved  material : 
through  a  porcelain  or  clay  filter  leaving  behind  the  bacteria  and 
other  undesirable  foreign  matter.     The  filtrate  could  be  tested 
for  the  possible  presence  of  such  bacteria  as  might  have  passed 
through  the  filter  and  these  destroyed  by  suitable  agents  (such  as 
will  not  interfere  with   the   activity   of   the  vaccine),   and   the 
filtrate  perhaps  concentrated  to  the  desired  degree  or  perhaps  used  j 
in  the  liquid  form.     However,  it  is  likely  that  the  present  method  of  ^ 
manufacture  and  use  of  the  smallpox  vaccine  will  continue  fori 
some  time.     The  marketed  smallpox  vaccine  should  contain  but ' 
few  viable  bacteria,  not  to  exceed  200  per  dry  point  or  per  gly- 
cerinated  tube.     According  to  extensive  tests  made  by  Rosenau : 
in   1 902-1 903,  dry  points  and  glycerinated  tubes  contained  asi 
high  as  44,000  bacteria  per  point  or  tube,  but  tests  made  since  J 
that  time  (Nelson  and  others)  show  much  lower  figures,  ranging] 
from   ten   or  fifteen  to  300  bacteria  per  point  or  tube.     Small-  ■ 
pox  virus  should  also  be  examined  (occasionally  at  least)  for  the| 
presence  of  colon  bacilli,   streptococci,  tubercle  bacilli  and  thej 
tetanus  bacillus.  1 

24.  Special  Biological  and  Toxicological  Tests 

Arsenic  in  Foods  and  Medicines — ^Biological  Test. — Arsenic 
is  widely  distributed  in  nature  and  is  extensively  used  in  the  arts 
and  industries.  Medicinally  it  is  a  very  popular  tonic  and  is  also 
much  used  as  an  insecticide  in  the  form  of  sprays  and  washes. 
Animal  hides  are  frequently  preserved  by  arsenic  which  account 
for  the  presence  of  this  poison  in  gelatin  made  from  such  hide 


SPECIAL  TOXICOLOGICAL  TESTS  269 

Fruits  and  vegetables  which  have  been  sprayed  with  arsenical 
compounds  for  the  purpose  of  destroying  insect  pests,  may  contain 
enough  of  this  substance  to  produce  symptoms  of  poisoning. 
Arsenic  is  occasionally  added  to  alcoholic  beverages  to  give  them  a 
tonic  effect.  It  has  been  demonstrated  that  very  minute  amounts 
of  arsenic  are  normally  present  in  various  organs  of  the  human 
body,  as  the  thyroid  gland,  thymus  gland  and  liver,  although 
some  investigators  question  the  correctness  of  this  claim.  However 
these  somewhat  problematical  traces  of  arsenic  in  organs  of  the 
human  body  and  also  in  the  organs  of  other  animals  need  not 
concern  the  food  and  drug  analyst  as  far  as  routine  work  is 
concerned. 

As  a  rule,  the  tests  for  arsenic  outlined  in  the  majority  of  text- 
books are  chemical  and  hence  this  work  is  usually  relegated  to  the 
chemical  laboratory.  Within  recent  years  attempts  have  been 
made  to  employ  biological  tests  for  determining  the  presence  of 
arsenic  in  food  substances,  based  upon  the  discovery  that  certain 
molds  when  growing  in  substances  containing  arsenic  will  give  rise 
to  garlic-like  odors. 

Gosio  demonstrated  that  certain  molds  which  when  grown  in 
and  upon  media  containing  very  minute  quantities  of  arsenic 
gave  rise  to  gaseous  compounds  characterized  by  a  garlic-like 
odor.  Seven  different  kinds  of  molds  have  this  power,  more 
especially  Penicillium  brevicaule,  which  Gosio  isolated  from  air 
and  which  he  frequently  found  on  decomposing  paper.  Crumbs 
of  bread  (wheaten)  form  the  culture  medium  for  this  mold  and 
the  incubation  is  done  at  28°  to  32°  C,  a  vigorous  growth  being 
produced  within  48  hr.  In  the  presence  of  not  more  than 
o.ooooi  gram  of  arsenic  in  such  culture  there  will  be  noticeable 
a  distinct  and  very  characteristic  garlicky  odor  which  may  persist ' 
for  months,  if  the  culture  is  not  killed.  These  arsenic  molds 
do  not  produce  garlic  odors  or  gases  with  sulphur,  phosphorus, 
antimony,  boron,  and  bismuth  compounds  but  they  do  have  the 
power   of    converting   selenium   and  tellurium   compounds   into 


270  BACTERIOLOGICAL  METHODS 

volatile  substances  having  the  garlic-like   odor.     The   following 
procedure  is  recommended. 

If  the  material  to  be  examined  is  Hquid,  let  the  dry  bread 
crumbs  (either  white  or  graham)  absorb  it  to  saturation,  and 
then  scatter  a  small  quantity  of  fine  crumbs  over  the  surface.  Tf 
the  material  to  be  tested  is  solid,  grind  or  cut  it  into  small  piece 
and  mix  with  an  equal  amount  of  the  bread  crumbs  and  then 
moisten  with  a  little  sterile  distilled  water.  Place  the  prepared 
material  in  sterile  flasks  of  suitable  size  and  plug  with  sterile 
cotton.  Sterilize  the  flask  and  contents  by  the  usual  fractional 
method  at  100°  C,  or  for  30  min.  in  the  autoclave.  Absolute 
sterilization  must  be  secured.  There  is  no  danger  of  volatilizing 
the  arsenic  at  these  temperatures.  As  soon  as  flask  and  contents 
are  cold,  inoculate  with  the  mold,  as  follows.  The  mold  culture- 
may  be  grown  on  bread  or  on  pieces  of  potato.  Remove  a  small 
quantity  of  the  mold  in  the  spore -forming  stage  and  mix  with 
peptone  salt  solution  or  sterilized  water.  Add  enough  of  thi^ 
mold  suspension  to  just  moisten  the  bread  in  the  flask.  Do  not 
add  more  of  the  spore-bearing  material  than  the  mass  (bread  and 
arsenical  substance)  in  the  flask  will  absorb  as  too  much  moistuii 
will  retard  growth.  Cover  the  inoculated  flask  with  a  rubber  cv. 
and  incubate  'at  a  temperature  of  37°  C,  although  the  ordinary 
room  temperature  will  answer  the  purpose.  As  soon  as  the  growth 
is  clearly  visible  to  the  naked  eye,  which  may  be  in  24  hr.,  the  char- 
acteristic garlic  odor  will  be  noticed  upon  opening  the  flask.  If 
no  odor  is  appreciable,  again  seal  and  incubate  for  another  24  hr. 
period  or  even  longer.  In  case  the  substances  to  be  tested  are 
strongly  acid,  they  may  first  be  neutralized  by  means  of  calcium 
carbonate.  It  must  also  be  kept  in  mind  that  Penicillium  brevi- 
caule,  as  well  as  other  molds,  will  convert  tellurium  and  selenium 
compounds  into  volatile  substances  having  a  garlic-like  odor.  The 
arsenic  and  tellurium  odors  are  very  closely  similar  but  that  from 
selenium  is  somewhat  different  in  quality,  more  like  that  of  mer- 
captan.     The  test  is  extremely  delicate,  o.ooooi  gram  of  arsenic 


SPECIAL  TOXICOLOGICAL   TESTS  27 1 

can  be  recognized  with  certainty.  A  solution  of  o.ooooi  gram 
of  potassium  tellurite  in  10  cc.  of  mold  infested  gelatin  medium 
in  a  cotton  plugged  test-tube  gave  out  a  strong  odor  of  garlic  for 
several  weeks. 

Biginelli  ascertained  that  the  gases  formed  by  Penicillium 
brevicaule  in  arsenical  cultures  were  completely  absorbed  by  solu- 
tions of  mercuric  chloride  with  the  formation  of  a  double  compound 
of  mercuric  chloride  and  diethyl  arsine  which  is  quite  easily  decom- 
posed accompanied  by  the  reappearance  of  the  garlic  odor. 

The  test  is  unlimited  in  its  application  and  will  respond  in  the 
presence  of  all  manner  of  organic  substances  and  bacterial  contami- 
nations. It  is  far  more  delicate  than  any  of  the  chemical  tests  and 
can  be  carried  out  in  much  shorter  time. 

Toxicity  Tests  with  Defibrinated  Blood. — The  older  physi- 
ologists and  toxicologists  made  the  interesting  observation  that  toxic 
substances  of  various  kinds  produced  certain  changes  in  the  blood. 
Some  poisons  disintegrated  the  red  corpuscles,  some  caused  the 
corpuscles  to  clump  or  to  agglutinate  and  still  others  reduced  or 
even  completely  inhibited  the  coagulating  power  of  the  blood. 
These  phenomena  have  suggested  the  possibility  of  estimating  or 
measuring  the  toxicity  of  certain  groups  or  classes  of  substances  by 
noting  the  effects  which  they  produce  when  brought  in  contact 
with  red  blood  corpuscles.  The  more  important  groups  of  toxic 
substances  which  give  rise  to  marked  reactions  with  red  blood 
corpuscles  are  the  toxalbumins  or  toxins,  the  saponins  and  many  of 
the  toxic  chemical  compounds.  The  following  tests  may  prove  of 
value  in  the  food  and  drugs  laboratories. 

Toxalbumins  or  Toxins. — Toxalbumins  and  toxins  are  poisonous 
substances  formed  in  plants  and  animals  as  the  result  of  microbic 
invasion  and  also  as  the  result  of  metabolism  in  the  plant  or  animal 
itself.  Of  special  interest  are  the  vegetable  toxalbumins  which 
possess  the  remarkable  property  of  clumping,  agglutinating  and 
finally  precipitating  red  blood  corpuscles  and  have  therefore  been 
designated  "vegetable  agglutinins."     A  mere  trace  of  these  sub- 


272  BACTERIOLOGICAL  METHODS 

stances,  when  added  to  defibrinated  blood  in  a  test-tube,  causes 
clumping  into  a  mass  resembling  sealing  wax.  The  most  im- 
portant vegetable  agglutinins  are  abrin,  ricin,  robin  and  crotin. 
Of  these,  ricin,  abrin  and  crotin  also  cause  the  coagulation  of  milk. 

To  make  the  agglutination  tests,  defibrinated  blood  is  used. 
Whip  the  fresh  blood  (of  ox,  horse,  guinea-pig  or  rabbit)  by  means 
of  twigs,  bunch  of  thin  wires,  wire  mesh  egg  beater,  or  run  the 
blood  into  Erlenmeyer  flasks  with  iron  filings  and  shake  vigorously 
for  several  minutes.  The  fibrin  is  deposited  on  the  twigs,  wires,  or 
on  the  iron  filings,  thus  separating  it  from  the  corpuscles  and  the 
serum.  Removing  the  serum  from  the  blood  and  displacing  it  by 
physiological  salt  solution  renders  the  reaction  more  pronounced, 
thus  pointing  to  the  existence  of  antiagglutinins  in  the  serum. 
Ricin  will  agglutinate  the  blood  of  the  guinea-pig  in  dilutions  of 
I  :  600,000.     Abrin,  crotin  and  robin  react  in  a  similar  manner. 

Saponins. — These  substances  are  widely  distributed  in  the 
plant  kingdom  and  have  chemical  properties  linking  them  with  the 
glucosides.  They  have  been  designated  nitrogen-free  glucosides. 
The  dry  powder  causes  violent  sneezing  when  inhaled  and  the 
aqueous  solutions  foam  when  shaken.  Most  of  them  are  neutral 
in  reaction  and  are  capable  of  holding  many  finely  divided  sub- 
stances in  suspension.  They  dialyze  with  diflSiculty  and  incom- 
pletely. They  dissolve  in  hot  as  well  as  in  cold  water  but  are  in- 
soluble in  absolute  alcohol  and  in  ether. 

Saponins  have  been  found  in  many  different  species  of  plants. 
The  more  important  and  better  known  are  digitonin  (in  Digitalis 
purpurea),  saponin  (Saponaria  officinalis),  githagin  {Agrosiemma 
githago),  senegin  {Poly gala  senega),  ssipomn  (Chlorogalum  pomeri- 
dianum),   struthiin    {Gypsophila  struthium),   sapotoxin    {Quillaja  \ 
saponaria    and    Sapindus    saponaria) ,    and    sarsaparilla-saponin  J 
(Sarsaparilla  species).     Saponins  are  highly  toxic  when  introduced  \ 
into  the  blood  directly  and  some  of  them  are  well-known  poisoning  \ 
agents.     American  Indians  have  long  made  use  of  the  roots  of 
Chlorogalum  for  the  purpose  of  stupefying  fish.     Most  saponins 


SPECIAL  TOXTCOLOGICAL  TESTS  273 

are  however  absorbed  quite  slowly  which  makes  it  possible  for  per- 
sons in  good  health  to  take  comparatively  large  quantities  of  weak 
solutions  without  producing  serious  harm.  They  are  protoplas- 
mic poisons  and  it  is  due  to  this  property  that  they  hemolyze  blood 
causing  it  to  become  laky.  It  has  been  demonstrated  experimen- 
tally that  saponins  act  more  energetically  upon  blood  corpuscles 
separated  from  the  serum  because  the  serum  contains  cholesterin 
which  retards  hemolysis.  It  is  suggested  that  the  hemolytic 
action  of  saponins  is  due  to  the  removal  of  the  Hning  membrane 
of  the  corpuscles  which  consists  of  lecithin,  forming  lecithin-sa- 
ponin.  Saponins  also  combine  with  cholesterin  (forming  choles- 
terin-saponin)  and  the  affinities  of  any  saponin  being  satisfied 
by  the  cholesterin,  it  no  longer  acts  upon  the  lecithin.  This  ex- 
plains why  cholesterin  retards  or  checks  the  hemolytic  action  of  the 
saponins.  The  saponins  also  dissolve  white  blood  corpuscles  but 
to  a  much  weaker  degree. 

In  making  the  blood  tests  for  the  presence  of  saponins,  isotonic 
(to  blood  serum)  or  physiological  salt  solution  (0.9  per  cent.)  is 
added  to  the  defibrinated  blood,  100  parts  to  one  of  the  defibrin- 
ated  blood.  Dilute  the  suspected  saponin  bearing  substance  with 
physiological  salt  solution  and  add  it  to  the  diluted  blood  suspen- 
sion. If  saponin  is  present  the  mixture  at  once  becomes  laky  due 
to  hemolysis.  Githagin  will  develop  the  hemolytic  action  in 
dilutions  of  i  :  50,000. 

It  must  be  borne  in  mind  that  a  variety  of  substances  will  pro- 
duce hemolysis,  such  as  ether,  chloroform,  alkalies,  gallic  acid  and 
solanine.  The  lytic  test  above  outlined  may  be  employed  as  a 
check  or  corroboration  of  the  chemical  and  perhaps  additional 
I  biological  tests. 

Chemical  Hemolysis. — Vandevelde  has  suggested  a  method  for 
determining  the  toxicity  of  chemical  compounds  by  hemolysis. 
Defibrinated  ox  blood  is  used  in  addition  to  the  following  solutions. 
A  solution  of  0.9  per  cent,  of  salt  in  50  per  cent,  alcohol  (by  volume, 
specific  gravity  of  0.9548  at  15°  C);  physiological  salt  solution 


274 


BACTERIOLOGICAL  METHODS 


(0.9  per  cent.)  and  a  suspension  of  5  per  cent,  defibrinated  ox 
blood  in  0.9  per  cent,  salt  solution. 

To  make  the  experiments  test-tubes  are  used  and  the  compound 
microscope  is  not  required.  In  a  series  of  standard  test-tubes 
place  2.5  cc.  of  the  suspended  blood  (in  the  sodium  chloride  solu- 
tion) and  the  same  amount  of  the  solution  to  be  tested  (in  varying 
amounts  of  physiological  salt  solutions,  therefore  different  strength 
solutions)  in  order  to  ascertain  the  exact  point  when  hemolysis 
takes  place.  A  solution  which  does  not  produce  hemolysis  after 
a  definite  period  of  time  (3  hr.),  but  which  does  result  in  hemolysis 
on  the  smallest  further  addition  of  the  substance  under  examina- 
tion is  spoken  of  as  a  ''critical  solution."  The  time  limit  in  these 
tests  is  3  hr.  If  after  the  expiration  of  this  period  of  time,  the  trace 
addition  of  the  solution  does  not  produce  the  hemolytic  effect, 
the  test  is  negative  and  the  next  stronger  or  higher  concentrate 
must  be  tried.  The  term  critical  coefficient  refers  to  the  number 
giving  the  concentration  of  the  substance  necessary  to  hemolyze  or 
kill  the  red  corpuscles. 

The  following  is  the  result  obtained  by  Vandevelde  regarding 
the  critical  solution  of  ethyl  alcohol. 


Cc.  of  Sus- 

Cc. of  NaCl  Sol. 

Cc.  NaCl 

i    Alcoholic  Sol.  in 

Reaction 

pended  Blood 

in  Alcohol 

Solution 

1  Volume  Per  Cent. 

1 

in  3  hr. 

2.5 

2.20 

0.30 

22.0 

Hemolysis 

2-5 

2.15 

0.35 

21.5 

Hemolysis 

2.5 

2.10 

0.40 

21.0 

Hemolysis 

2.5 

2.05 

0.45 

20. 5 

Hemolysis 

2.5 

2.00 

0.50 

j           20.0 

Hemolysis 

2.5 

1.95 

0.55 

1           19-5 

Negative 

2.5 

1.90 

0.60 

i           19-0 

Negative 

From  this  table  it  will  appear  that  the  critical  solution  of  ethyl 
alcohol  contains  19.5  cc.  of  absolute  alcohol  in  100  cc,  or  15489 
grams  of  alcohol  in  100  cc.  According  to  Vandevelde,  the  addi- 
tion of  methyl  alcohol  diminished  the  toxicity  of  ethyl  alcohol, 
whereas  the  higher  alcohols  were  found  to  be  more  toxic  than  the 


SPECIAL  TOXICOLOGICAL  TESTS  275 

latter.  Giving  the  toxicity  of  loo  parts  of  ethyl  alcohol  as  loo, 
then  47  parts  by  weight  of  isopropylic  alcohol,  29  parts  of 
^sobutylic  alcohol  and  12.5  parts  of  amy  lie  alcohol  were  found  to 
De  isotoxic  with  that  quantity  of  ethyl  alcohol. 

The  term  toxin,  more  accurately  speaking,  appHes  to  poisonous 
substances  elaborated  by  bacteria  and  which  require  an  incuba- 
:ion  period  before  forming  antibodies  or  antitoxins.  The  toxins 
:ormed  by  the  bacterial  group  appear  to  be  intimately  associated 
A^ith  the  life  processes  of  the  living  cell,  but  their  chemical  com- 
position remains  thus  far  unknown.  We  know  that  they  are  very 
•eadily  destroyed  by  heating  (60°  to  80°  C.)  and  that  they  are 
chemically  very  unstable,  and  that  they  are  among  the  most  highly 
poisonous  agents  known  to  science.  They  are  far  more  toxic  than 
:he  potent  vegetable  alkaloids  and  animal  toxalbumins,  as  is  shown 
n  the  following  tabulation  (Jordan): 

Atropine,  fatal  dose  to  man 130  mg. 

Strychnine,  fatal  dose  to  man 30-40  mg. 

Cobra  venom,  fatal  dose  to  man 4-375  mg- 

Tetanus  toxin,  fatal  dose  to  man o.  23    mg. 

Various  animals  produce  toxalbumins  or  toxins,  as  snakes 
crotalin,  viperine),  scorpions,  tarantulas,  the  Gila  monster  and 
ther  lizards.  Rattle-snake  venom  evidently  possesses  a  variety 
f  properties.  It  will  agglutinate  blood,  neutraHze  the  fibrinogen, 
emolyze  red  corpuscles,  and  is  highly  neurotoxic.  Within  recent 
ears  antibodies  have  been  produced  against  these  several  toxic 
Libstances. 

Muscarine,  the  toxic  agent  of  Amanita  muscaria  (fly  agaric),  is 
n  alkaloid  which  acts  very  quickly,  whereas  the  toxic  agents  of 
manita  phalloides  and  A.  verna  are  toxin-like  in  that  there  is  an 
icubation  period  of  from  10  to  14  hr.  before  the  toxic  symptoms 
egin  to  manifest  themselves.  They  are  strongly  hemolytic.  It 
supposed  that  the  pollen  grains  of  certain  flowers  contain  toxin- 
ke  substances  to  which  certain  persons  are  pecuHarly  suscepti- 
e.  All  toxalbumins  or  toxins,  whether  derived  from  bacteria, 
19 


276  BACTERIOLOGICAL   METHODS 

fungi  or  higher  plants,  possess  the  very  characteristic  property  of 
forming  antibodies  or  antitoxins.  Thus  there  is  antivenin  used  in 
the  treatment  of  snake-bite,  also  antibodies  used  in  the  treatment 
of  hay  fever,  etc.,  which  products  are  more  fully  described  in  works 
on  medical  bacteriology. 

Frog  Tests  for  the  Presence  of  Alkaloids. — ^Lively  small  frogs 
respond  quite  readily  to  the  action  of  even  high  dilutions  or  very^ 
minute  quantities  of  vegetable  alkaloids.  It  is  suggested  that 
the  food  bacteriologists  perform  the  following  tests,  which  are 
frequently  desired  as  a  check  or  corroboration  of  the  findings  of 
the  chemist  and  toxicologist. 

The  material  used  for  the  frog  injections  is  the  evaporated 
ether  extract  dissolved  in  a  small  amount  of  sterilized  distilU^i 
water.     The  injections  are  made  hypodermically  in  the  lymph  s 
on  the  back  of  the  frog.     It  is  advised  that  the  tests  be  made  in 
duplicate  and  repeated  as  often  as  may  be  required  to  attain  ab- 
solutely conclusive  results.     Each  test  should  be  checked  by  in- 
jecting approximately  minimal  fatal  doses  of  the  pure  alkaloid  it- 
self, obtained  from  some  reliable  house.     This  will  make  it  p' 
sible  to  note  the  toxic  symptoms  produced  by  the  pure  alkaloid,  i\  1 
compare  with  the  symptoms  produced  by  the  suspected  alkaloid 
the  substance  under  consideration.     These  check  tests  are  gene 
ally  omitted  by  the  analyst  who  has  had  extensive  laboratory  < 
perience  and  who  is  therefore  in  a  position  to  recognize  the  nati: 
of  the  poison  (alkaloid)  from  the  symptoms  manifested  by  1 
inoculated  frog.     In  many  instances  the  frog  alkaloidal  tests  m 
serve  as  checks  upon  the  blood  tests  already  described. 

In  extracting  the  suspected  substances  it  must  be  kept  in  mii 
that  alkaloids  are  very  sparingly  soluble  in  water,  but  when  acidu- 
lated (hydrochloric  acid  about  i  per  cent.)  water  is  used  the  a< 
forms  the  salt  (chloride)  which  is  readily  soluble  in  water.     Alk.. 
loids  are  soluble  in  ether  and  in  mixtures  of  ether  and  chloroform, 
therefore  these  reagents  should  be  used  rather  than  the  acidulatt  d 
water,  especially  since  they  are  also  antiseptic  and  the  extracti\ 


SPECIAL  TOXICOLOGICAL  TESTS  277 

which  they  yield  are  freer  from  extraneous  impurities,  and  also 
because  the  residue  which  is  injected  into  the  frog  (dissolved  in  the 
water)  represents  the  alkaloid  and  not  its  chloride.  Should, 
however,  the  evaporated  acidulous  extract  be  used  for  making  the 
frog  test,  then  the  check  test  should  be  made  with  the  correspond- 
ing pure  alkaloidal  salt. 

If  the  inoculated  frog  shows  no  marked  symptoms  of  poisoning 
in  the  course  of  3  or  4  hr.,  it  is  very  likely  that  the  residue  under  sus- 
picion does  not  contain  any  very  poisonous  substance.  The  pri- 
mary object  of  the  tests  is  to  ascertain  whether  or  not  dangerously 
toxic  substances  (alkaloids)  are  present  in  foods,  and  not  for  the 
Durpose  of  determining  the  identity  of  the  alkaloid  nor  to  demon- 
trate  the  presence  of  comparatively  nontoxic  alkaloids. 


INDEX 


Abrin,  272 
Abscesses,  201 
Acne,  201 
Actinomyces,  184 
Adjustment  of  media,  74 
Agar,  Hesse's,  135 

litmus,  78 

nutrient,  77 

test,  14 
Agglutination,  108 
Agglutinins,  vegetable,  271 
Albumen  coagulation,  252 
Alcohol,  212 
Aldehydes,  216 
Algae,  14 

in  water,  114 
Alkaloid  tests,  276 
Amanita  muscaria,  275 

phalloides,  275 

vema,  275 
Ampuls,  202 
Analytical  reports,  17 
Anderson-McClintic,  231 
Animal  fat,  161 
Animals,  diseased,  26 
Antiformin,  135 

methods,  135 
Antimony  test,  16 
Apparatus,  bact.,  83 

counting,  41 
Aquae,  198 
Arachnodiscus,  14 
Arrak,  225 
Arsenic.  268 


Arsenical  test,  15,  268 
Ash  determination,  10 
Aspergillus,  224,  227 
Atropine,  198,  275 
Autoclave,  73 
Azolitrnin,  74 

B 

Baby  food,  5 

Bacillus  aerogenes,  132,  155 

B.  anthracis,  184 

bifermentens,  153 
botulinus,  158,  177,  183 
bulgaricus,  139 
calif orniensis,  206 
caucasica,  226 
cholerae,  112 

test  for,  113 
coli,  91,  94 

in  milk,  128 
cyanogenes,  132 
enteritidis,  loi,  158 
erythrogenes,  132 
gelatinosum,  205 
gummosus,  205 
kiitzingianum,  214 
levaniformans,  203 
liodermos,  203 
mesentericus,  203 
oxydans,  214 
paratyphosus,  102 
pasteurianum,  214 
perfringens,  153 
prodigiosus,  132 
psittacosis,  102 
soja,  227 


279 


28o 


INDEX 


B.  subtilis,  127,  153 

suipestifer,  102 

synxanthus,  132 

tetani,  180,  183,  199 

tuberculosis,  133,  136 

typhi  murium,  loi 

typhosus,  102 
in  water,  105 

vermiforme,  228 

viscosus,  219 

vulgatus,  203 

Welchii,  155 

xylenum,  230 
Bacon  beetle,  140 
Bacteria,  23 

dead,  49,  50 

examination  of,  13,  35 

in  foods,  23,  58 

in  cream,  132 

in  ice  creams,  138 

in  meats,  183,  153 

in  miUc,  123 

in  water,  117 

intestinal,  91 

lactic  acid,  63,  140 

living,  49,  50 

of  cadaver,  184 

of  eggs,  195 

of  vinegar,  229 

potato  group,  203 

sugar,  203 
Bacterial  dilutions,  86 
Bacterins,  265 
Bacteriological  reports,  20 

technique,  83 
Bark  tissues,  PI.  VI 
Bartow,  E.,  25 
Bast  cells,  PI.  II 
Beaker  sand  test,  10 
Bean  tissues,  PI.  V 
Beebe  wine,  229 
Beef  fat,  164 
Beer,  218 

diseases,  219 


Beer,  ginger,  228 
Beetle,  bacon,  140 
Belladonna,  4 
Benzoic  acid  test,  11 
Berries,  polluted,  26 
Biginelli,  271 
Bile,  93 

lactose,  79 
Binders,  meat,  180 
Biological  products,  265 
Bismuth  test,  16 
Bitter  beer,  221 

cheese,  141 

milk,  132,  141 
Bitting,  51 
Black  cheese,  141 
Blanks,  report,  18 
Blood  tests,  271 
Blue  cheese,  141 

milk,  132 
Body  cell  count,  125 
Body  cells,  in  milk,  124 
Boehme,  100 
Boiled  milk,  131 
Boils,  201 
Boric  acid  test,  11 
Botulism,  177 
Bouquet,  217 
Brandy,  216 

rectified,  217 
Brautegam,  167 
Bread  contamination,  25 
Breed,  125 
Broca,  G.,  49 
Broth,  dextrose,  77 

liver,  79 

nutrient,  76 
Broths,  sugar,  77 
Buckwheat,  PI.  Ill 
Biirker  ruling,  45 
Butter,  167 

fat,  121 

renovated,  126 
Buttermilk,  139 


INDEX 


281 


Cadaver  bacilli,  1 84 
Candies,  209 
Canned  foods,  63 

meats,  154 
Carbuncles,  201 
Carriers,  typhoid,  27,  103 
Cassia  buds,  PI.  IV 
Castor  oil,  160 
Catsups,  54,  59 
Centrifugal  tube,  38 
Cestoda,  ova,  67 
Cheese,  54 

bitter,  141 

black,  141 

blue,  141 

hopper,  140 

mite,  140 

Penicillium,  143 

poisoning,  142 

putrid,  141 

ripening,  140 

skipper,  140 

spoiling  of,  140 
Chemical  hemolysis,  273 
Chemists,  i 
Chinese  eggs,  191 

gardeners,  25 
Chlorogalum,  272 
Cholera,  Asiatic,  112 

germ  test,  113 
Cholesterol,  161 
Cider,  hard,  230 
Cinchona  test,  9 
Citromyces  glaber,  215 

pfefferianus,  215 
Cladosporum,  197 
Clams,  151 

Clove  stems,  PI.  Ill"  - 
Coagulation  coefficient,  252 
Cobra  venom,  275 
Cocaine,  198 
Coffee  adulterants,  PL  IV 


Cold  storage  eggs,  191 
Colon  bacillus  in  water,  117 
test,  97 

typhoid  bact.,  94 
Color  reactions,  10,  15,  160 
Colors,  of  oils,  160 
Concentrates,  35,  38 
Condensed  milk,  143 
Condiments,  209 
Congeners,  216 
Conium  test,  9 
Conn's  bacillus,  141 
Contamination,  91 

limits,  52 

sewage,  97 
Copper  test,  16 
Corn  syrup,  208 

tissues,  PI.  V 
Counting  apparatus,  41 
Counts,  44 
Coupin,  263 
Cream,  bacteria  in,  132 

ripening,  54,  132 
Crotalin,  275 
Crotin,  272 
Crystals,  PL  II 

fat,  159, 
Cultural  methods,  68 
Culture  media,  69,  71 

standard,  75 
Cultures,  mixing  of,  85 

plate,  85 

tube,  87 

types,  87,  89 
Curcuma  thread,  1 1 

D 

Davis,  50 

Deposits,  finger  nail,  201 
Dematium  pullulans,  219 
Desmids,  116 
Dextrose  broth,  77 
Diatoms,  14 


282 


INDEX 


Dilutions,  43 

bacterial,  86 
Dioxide  test,  129 
Diplococcus,  of  eggs,  191 
Direct  examination,  36 
Dirt,  ID 
Diseases,  26 
Disinfectants,  230,  259 
Distillation,  217 
Dried  eggs,  190 
Drigalski,  no 
Drinks,  fermented,  210 

medicated,  218 
Drugs,  classification,  2 
Dusting  powders,  201 


Eber's  test,  179 

Edelmann,  167 

Eels,  vinegar,  55 

Efficiency,  of  disinfectants,  258 

Egg  bacteria,  195 

decomposition,  196 

media,  195 

membrane,  189 

tests,  188 
Eggs,  187 

Chinese,  191 

dried,  190 

examination  of,  192 

frozen,  191 

storage,  191 
Ehrlich,  100 
Ellis,  153 
Emery,  J.  A.,  164 
Emich,  F.,  15 
Endo  medium,  80 
Enzymes,  93 
Equipment,  6 
Ergot,  198 
Essential  oils,  263 
Estimates,  quantitative,  4 
Evaporated  eggs,  190 


Evaporation,  40 
Examination,  direct,  35 

of  eggs,  193 
Excrement,  human,  25 


Factory  methods,  61 
Fat  crystals,  159 

in  milk,  121 

tests,  127 
Feces,  human,  91 
Fermentation,  212 
Fermented  drinks,  210 

foods,  210 
Ferments,  acid  forming,  214 
Fertilizer,  human,  25 
Fillers,  ice  cream,  138 

meat,  180 
Filter,  clay,  126 
Filtering,  36 
Filtration,  fractional,  39 
Finger-nail  deposits,  201 
Fish  meats,  155 

pickled,  158 
Fitzgerald,  170 
Flies,  61 

Fluid  extracts,  197 
Food,  contamination,  23 

fermented,  210 

laboratory,  32 

poisons,  29 
Foods,  classification,  2,  3 

investigation,  32 

polluted,  25 
Foot-and-mouth  disease,  139 
Formaldehyde  test,  12 
Fornet,  170 
Frog  tests,  276 
Frozen  eggs,  191 

milk,  144 
Fruit  fresh,  52 

juices,  203 

rotten,  64 


INDEX 


283 


Fruit,  starch,  12 

whole,  52 
Fruits,  acid,  62 
Fusel  oil,  216 


Gaertner  bacilli,  loi,  158 
Gas  formation,  98^ 
Gay,  170 
Geinitz,  263 
Gelatin,  tests  for,  72 

moldy,  65 

nutrient,  77 
Gila  monster,  275 
Ginger  beer,  228 
Githagin,  273 
Glassware,  70 
Globules,  fat,  121 
Gluten  tests,  13 
Glycerin,  198 
Glycine  hispida,  227 
Gmelin,  50 
Gold  test,  16 
Goose  fat,  160 
iGosio,  50 
jGraham,  181 
jGrahe's  test,  9 
Grape  sugar,  167 
Greenlee,  190 
Gum  formers,  203 

H 


Hiss'  medium,  79 
Hog  cholera,  184 

bacteria,  loi,  158 
Hopper,  of  cheese,  140 
Hops,  218 
Horse  meat,  167 
Housewife,  52 
Howard,  B.  J.,  46,  48 
Howell,  Miss  K.,  25 
Hydrogen  dioxide  test,  129 
Hygienic  lab.,  32 
Hypodermic  syringes,  174 


Ice-cream  bacteria,  138 

fillers,  138 
Ice  creams,  138 
Incubation,  88 
Indol  test,  1 00 
Infections,  24 

skin,  201 
Intestinal  bacteria,  91 
Iodine  reaction,  12 
Iron  test,  16 
Itch  mite,  201 


Jams,  58 
Jordan,  275 


K 


Hamilton,  H.  C.,  244 
Hand  gluten  test,  13 
JHanford  epidemic,  27 
Hard  cider,  230 
Hansen,  50,  213 
Heat  sterilization,  26 
Hemacytometer,  36,  43 
Hemolysis,  chemical,  273 
Herring,  pickled,  57 
Hesse's  agar,  135 


Kebler,  L.  F.,  229 
Kerr,  R.  H.,  161 
Kephir,  226 
King,  50 

Kitasato  filter,  37 
Knapp,  131 
Koch,  Robert,  113 

cholera  test,  113 
Koenig,  17 
Koumiss,  227 


INDEX 


Laboratory,  equipment,  6 

food,  I 

methods,  2,  30,  31 
Lactose  bile,  79 

litmus  agar,  78 
Lafar,  204 
Lancet  method,  230 
Lard,  164 

oil,  160 
Lead  test,  16 
Leaks,  62 
Leban, 228 
Leuconostoc,  205 
Levan,  205 
Lignin  test,  9 
Limits,  of  contam.,  62 

of  organisms,  51 
Linseed  oil,  160 
Liver  broth,  79 
Lloyd,  J.  U.,  229 
Loop,  platinum,  85 

tubes,  no 
Lumpy  jaw,  184 

M 

Mace  test,  9 
Magpotine,  119 
Mallow  leaf,  PI.  V 
Manufacturers,  52 
Martindale,  263 
Maya,  226 
Meat  bacteriology,  177 

binders,  180 

canned,  154 

extracts,  69 

fillers,  180 

horse,  167 

of  fish,  155 

organisms,  181 

precipitins,  169 

sausage,  177 


Meat,  spoiled,  154,  174 

starch  in,  181 

toxins,  175 
Meats,  152 

dried,  157 

smoked,  157 

storage,  155 

sugar  in,  167 
Media,  reaction  of,  74 

standard,  75 

tubing  of,  84 
Medicamenta,  198 
Medicated  drinks,  218 
Medicinal  syrups,  203 
Medicines,  197 
Mercury  test,  16 
Methods,  bacteriological,  96 

cultural,  68 

laboratory,  2,  30,  32 

tabulation,  31 
Metschnikoff,  29 
Meyer,  Karl  F.,  169 
Micro-analysts,  i,  3,  6 
Micrococcus  casei  amari,  141 
Micro-gluten  test,  13 
Microscope,  i,  6 
Microscopical  reports,  18 
Miescher's  bodies,  187 
Milk,  26 

artificial,  82 

bacteria  in,  124 

bitter,  132,  141 

blue,  132 

boiled,  131 

body  cells,  124 

condensed,  143 

dried,  144 

examination  of,  120 

fat  count,  122 
globules,  121 

frozen,  144 

medium,  81 

powdered,  144 

raw,  131 


INDEX 


285 


Milk,  red,  132 
ropy,  132 
sour,  139 
standards,  123 
tuberculous,  128 
water  in,  131 
yellow,  132 

Mineral  waters,  118 

Miquel,  117 

Mite,  of  cheese,  140 

Molasses,  203,  208 

Mold  counting,  47 
in  foods,  58 
in  gelatin,  65 
in  plums,  56 
in  tomato  pulp,  51 

Morphine,  198 

Mucor,  204 

mucedo,  211 

Muller,  170 

Mussels,  151 

Mycoderma,  204,  214 

N 

Nelson,  268 
Nematodes,  61,  66,  69 
Neutral  red  test,  100 
Nostoc,  114 
Novy,  29 
Nutrient  agar,  77 

broth,  76 

gelatin,  77 
Nutrose,  82 

media,  135 


Oberhefe,  213 
Ohno,  T.,  244 
Oidium  lactis,  127,  147 
Oils,  essential,  263 
Oleomargarine  test,  126 


Olive  pits,  PI.  Ill 
Organisms,  identifi.,  54 

in  food,  58 

in  meat,  182 
Organoleptic  tests,  58 
Oscillaria,  114 
Ova,  66 
Oysters,  144 

shucked,  151 


Pancake  flour,  5 
Pancreatin,  93 
Paramecia,  116 
Parasites,  14 

intestinal,  66 
Pastes,  tomato,  59 
Pear  pulp,  PI.  II 
Penicillium  brevicaule,  269 

glaucum,  140,  142 
Peptone  medium,  82 
Percentage  estimates,  4 
Petri  dishes,  83 
Pfeiflfer's  phenomenon,  113 
Pharmaceutical  laboratory,  200 

sanitation,  200 
Pharmaceuticals,  197 
Phenol  coefficient,  233,  244 
Phenolphthalein,  74 
Phloroglucin,  9 
Phytosterol,  161 
Phytophthora,  197 
Pickles,  54 
Pilocarpine,  198 
Pine  tissues,  PI.  Ill 
Pink  colonies,  99 
Piophila  casei,  140 
Planting  in  media,  85 
Plate  cultures,  85 
Platinum  loop,  85 
Podkoassa,  226 
Poisons,  of  foods,  29 


286 


INDEX 


Pollen  grains,  PI.  I 
Pollution,  of  foods,  26 
Potassium  hydroxide,  9 
Powdered  milk,  144 
Powders,  201 
Precipitin  meat  test,  168 
Prescott,  105 
Preserves,  58 
Products,  canned,  63 
Proteus  vulgaris,  153 
Pseudo-trichinae,  187 
Pulp,  decomposed,  48 

mold,  51 

tomato,  46 
Pus  cells,  in  milk,  125 

organisms,  198 
Putrid  cheese,  141 

Q 

Qualitative  determinations,  90 
Quantitative  methods,  68 


Rating,  of  shellfish,  148 
Rattle-snake,  275 
Ravenel,  63 
Raw  milk  test,  131 
Reaction  limits,  17 

of  media,  74 
Red  milk,  132 
Report  blanks,  18 
Reports,  analytical,  17 
Rhamnus  bark,  PL  VI 
Rhizopus,  197 
Rice  tissues,  PI.  IV 

wine,  223 
Ricin,  272 
Rideal- Walker,  230 
Ripening,  of  cream,  132 
Robin,  272 
Ropiness,  of  beer,  219 


Ropy  milk,  132 
Rotten  fruit,  64 


Saccharomyces  species,  213 

anomalus,  215 

ellipsoides,  216 

hansenii,  215 

mycoderma,  214 

pasteurianus,  215 

pastorianus,  221 

ruber,  141 

sak6,  225 

soja,  227 
Sak6,  223 

Salicylic  acid  test,  1 1 
Sand, 4 

test,  ID 
Saponins,  272 
Sarcina,  228 

hamayuchia,  227 
Sarcocystis,  187 
Sauerkraut,  54 
Sausages,  177 
Savage,  158 
Sawyer,  W.  A.,  27 
Scalp,  201 

lotions,  201 
Schneider,  A.,  232 
Sclerenchyma  cells,  PL  II 
Sea  water,  95 
Selenium,  269 

test,  49 
Sera,  265 
Sewage,  91 
Shellfish,  144 
Siedentopf,  13 
Silver  test,  16 
Skin  infections,  201 
Skipper,  of  cheese,  140 
Slant  cultures,  89 
Smallpox  vaccines,  268 
Soda  fountain,  202 


INDEX 


287 


Soda  bottling,  207 
Sodium  caseinate,  82 
Soja  bean,  227 

sauce,  227 
Soup  stocks,  154 
Sour  milk,  139 
Souring  of  beer,  220 
Spaghetti,  27 
Spiritus  f rumen ti,  216 
Spoiled  meats,  174 
Spore  counter,  41 
Spores,  in  catsups,  53 

in  pepper,  57 
Sputum,  136 
Stab  cultures,  87 
Standard  media,  75 
Standardization,  of  milk,  123 
Starch,  in  fruits,  12 

filler,  12 

in  meat,  168,  181 

paper,  12 

test,  12 
Starches,  PL  I 
Starkey,  no 
Sterilization,  of  foods,  26 

of  media,  73 
Stiles,  G.  W.,  33 
Stitt,  137 

Storage  meats,  155 
Streptococcus  acidi  lactici,  127 
'  aureus,  130 

hollandicus,  132 

lacticus,  132 

longus,  153 

pyogenes,  130 
Strychnine,  275 
Sublimation  tests,  11 
Sugar,  in  meats,  167 

broths,  77 

grape,  167 

test,  in  meat,  167 
Sugars,  invert,  203 
Sulphurous  acid  test,  12 
Syringes,  hypodermic,  174 
Syrup,  corn,  208 


Syrups,  198,  202 

medicinal,  203 
Swells.  62 


Technique,  bact,,  83 
Tellurium,  269 

test,  49 
Temperature  differential,  99 
Tests,  blood,  271 

quantitative,  36 

tubes,  83 
Tinctures,  197 
Tomato  pastes,  59 

pulp,  46 
Tonsillitis,  26 
Torula,  204 

amara,  132 
Toxalbumins,  271 
Toxicity  coefficient,  249 
Toxins,  271 

in  meat,  175 
Treacle,  203 
Trematodes,  66 
Trichinae,  184 
Trichomes,  PI.  V 
Tube  cultures,  87 
Trioglyphis  siro,  140 
Tube,  centrifugal,  37 
Tuberculous  milk,  128 
Tubes,  loop,  no 
Tubing  media,  84 
Turbid  beer,  221 
Turck  ruling,  36,  44 
Typhoid  bacillus,  102 

carrier,  27,  103 

epidemics,  106 

infection,  25 

medium,  79 

methods,  105 

U 

Ultra-microscope,  13 
Unsanitary  methods,  61 


288 


INDEX 


Vaccines,  265 
Vacuum,  partial,  40 
Vandevelde,  274 
Vaughan,  29,  138 
Vegetable  fat,  161 
Vinegar  bacteria,  229 

eels,  55 
Viperine,  275 


Whiskey,  216 

rectified,  217 

Widal  test,  107 

Wilson,  105 

Wine,  bebee,  229 
slimy,  227 

Wines,  222 

diseased,  222 


W 

Water,  analysis  of,  114 

bacteria,  117 

bottled,  118 

mineral,  118 

tests,  114 
Watered  milk,  131 
Waters,  polluted,  25 
Weigmann's  bacillus,  14] 
Wheat  flour,  5 

tissues,  PI.  IV 


Yeast,  upper,  213 
Yeasts,  204 

in  foods,  58 
Yellow  milk,  132 
Yoghurt,  226 


Zappert  ruling,  44 
Zinc  test,  16 
Zsygmondy,  13 
Zymases,  212 


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